ANALYTICALBIOCHEMISTRY
188,53-64
(1990)
A Kinetic Method for Determination of Free Vanadium(W) and (V) at Trace Level Concentrations D. C. Crans,l
M. Shaia Gottlieb,
Department
of Chemistry,
Received
17,1989
July
Colorado
J. Tawara,
State University,
R. L. Bunch,
A kinetic method based on alkaline phosphatase has been developed to measure free trace levels of vanadium(IV) and (V). The method involves measuring the rate of the alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate with (VJ and without (V,) a competitive inhibitor in the assay. Michaelis-Menten kinetics for a competitive inhibitor was used to express the relationship between Vo/Vi and the inhibitor concentration. Measuring both V,-, and Vi thus yields a Vo/ Vi ratio that allows calculation of the competitive inhibitor concentration. Determination of free vanadium in complex fluids can be accomplished by comparing the ratio of rates of p-nitrophenyl phosphate hydrolysis with and without a sequestering agent to the ratios of rates measured on addition of a known vanadium concentration. Free vanadium(V) can conveniently be measured from lo-’ to lo-’ M and free vanadium(IV) can be measured at 10-s M and above. The error limits on the vanadium determinations range from +3 to +-12% of the concentration under investigation depending on the conditions under which the assay was conducted. 0 1990 Academic Press. Inc.
Vanadium is an important dietary trace element (1,2). The major sources of vanadium intake in mammals come from seeds,grains, roots, and water. Vanadium has a number of biological effects, including normalizing glucose levels and stimulation of hexose transport. It is an insulin mimetic agent, and it affects the cardiac muscle in mammals (1,2). It is a potent inhibitor for ribonucleases, ATPases, nucleases, and phosphatases and is a required cofactor in some nitrogenases (3,4) and bromoperoxidases (5-8). Organic vanadate derivatives can be substrates for glycolytic and pentose phosphate shunt enzymes (9-11). Vanadium(IV) is the major form pres1 To whom 0003.2697/90 Copyright All rights
correspondence
should
$3.00 0 1990 by Academic Press, of reproduction in any form
be addressed.
and L. A. Theisen
Fort Collins, Colorado
80523
ent in mammals. Tunicates have also been found to contain vanadium(II1) (12). Available analytical techniques can measure total vanadium or vanadium in oxidation states III, IV, or V (13). Oxidation states IV and V interconvert readily under physiological conditions, and it is often a problem to determine the active vanadium species in biological studies (1,2). Consequently, we present an enzymatic method that will measure free vanadium(IV) and (V) simultaneously in the concentration range from 10m5to lo-’ M vanadium. Analytical methods currently used to determine trace levels of vanadium include atomic absorption spectrometry (AAS’; detection range, parts per billion), neutron activation analysis (NAA; detection limit, 10-l’ M), inductively coupled plasma (ICP; detection range, from 10e3 to 10e6 g/g), dc plasma atomic emission spectrophotometry (DCPAES; detection range, from lop6 to 3 X lo-’ g/ml), mass spectrometry (detection range, parts per million), electron paramagnetic resonance spectroscopy (EPR; detection range, from lop5 to 1O-4 M), and nuclear magnetic resonance (NMR; detection limit, 1O-5 M) (12). With the exception of EPR and NMR spectroscopy, the above methods measure total vanadium con-
’ Abbreviations: AAS, atomic absorption spectrometry; NAA, neutron activation analysis; ICP, inductively coupled plasma; DCPAES, dc plasma atomic emission spectrophotometry; EPR, electron paramagnetic resonance spectroscopy; BTP or Bis-Tris propane, 1,3-bis[(tris(hydroxymethyl)methyl)amino]propane; EPPS, iV’-(2hydroxyethyl)-N-piperazinepropanesulfonic acid; TAPS, 3-[(tris(hydroxymethyl)methyl)amino]propanesulfonic acid, TES, 2[(tris(hydroxymethyl)methyl)amino]ethanesulfonic acid; PIPES, piperazine-N,N’-b&(2-ethanesulfonic acid); DEA, diethanolamine; Bicine, N,N-bis(2-hydroxyethyl)glycine; NEM, N-ethylmorpholine, MDEA, N-methyldiethanolamine; TEA, triethanolamine; AMPSO, 2-[(l,l-dimethyl-2-hydroxyethyl)amino]ethanesulfonic acid; DIPSO, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid; IEA, triisopropanolamine; TAPSO, 3-[N-(tris(hydroxymethyl)methyl)amino]-2-hydroxypropanesulfonic acid; Tricine, N-[tris(hydroxymethyl)methyl]glycine; Bis-Tris, [bis(2-hydroxyethyl)amino]tris[(hydroxymethyl)methane]; MIDA, N-methyliminodiacetic acid; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid. 53
Inc. reserved.
54
CRANS
ET
AL.
centrations and do not distinguish between different vanadium species or oxidation states. EPR will detect only V(IV) species and 51V NMR will detect only V(V) species. Studies requiring determination of both V(IV) and V(V) concentrations therefore involve experiments using both EPR and 51V NMR. Although such studies have been conducted in the past, the low sensitivity, the nonroutine instrumental requirements of both EPR and NMR, and the nonroutine use of EPR make such studies difficult to coordinate and quantitate (1,2,13). Biological studies of vanadium would benefit from an experimentally simple quantitative technique. Kinetic methods have previously been used to determine trace level organic compounds, inorganic compounds, and proteins in biological fluids (14-16). We present a kinetic method based on the alkaline phosphatase catalyzed hydrolysis of p-nitrophenyl phosphate to form p-nitrophenol and phosphate. Alkaline phosphatase hydrolyzes p-nitrophenyl phosphate with an uninhibited rate, VO; however, in the presence of a competitive inhibitor, the inhibited rate, Vi, will reflect the concentration of inhibitor. Using Michaelis-Menten kinetics, a direct relationship can be derived between the competitive inhibitor concentration [IleE and the V,,/ Vi ratio (17-20). Alkaline phosphatase is competitively inhibited by phosphate and phosphate analogs including arsenate, vanadate, and molybdate (17,18,21-24). Vanadate is a much more potent inhibitor (Ki - 10e6 M) than phosphate (Ki - 10e3 M), possibly because a trigonal-bipyramidal vanadate-enzyme complex (transition state analog) is formed (1,21-24). Vanadyl cation is also a potent competitive inhibitor for alkaline phosphatase. The hydrolysis rates of p-nitrophenyl phosphate are therefore a sensitive measure for low concentrations of vanadate and vanadyl cation. This approach has been developed to measure concentrations of free vanadate and/or vanadyl cation (simultaneously) at lo-’ to 1O-5 M levels in complex samples.
stock solution was prepared by dissolving appropriately weighed vanadium pentoxide (V,OJ with 2 eq of sodium hydroxide to generate a vanadate solution of approximately 0.25 M; this solution was stored at 4°C. The vanadyl cation stock solution was prepared by dissolving appropriately weighed vanadyl sulfate (VOS04.H20) into 0.1 M H2S04 to generate a vanadyl sulfate solution of approximately 0.5 M (25). No changes in the concentrations of the vanadate or vanadyl cation standard solutions, which were monitored over the course of 6 months, were seen. The exact concentration of the vanadate stock solution was determined by diluting the solution lOOO-fold into 0.1 M NaOH and measuring the absorbance at 260 nm. The major (“only”) vanadate species in this solution is HVOi- , and by using an extinction coefficient of 3.55 X lo3 M-l cm-’ the concentration of the diluted stock and original stock could be calculated (27). At no time was acid added to a solution containing vanadate, since vanadate in the presence of acid generates the orange decamer (1,2,28). The exact concentration of the vanadyl stock solution was determined by measuring the absorbance of this solution at 750 nm. The concentration was then calculated using a molar extinction coefficient of E= 18.0 -t 0.2 M-l cm-’ (25). Alternatively, the concentration of the vanadyl cation stock solution could be determined using a permanganate-oxalate titration procedure (26). The stock solutions were kept at 4°C. Since the pH < 2, we observed no oxidation of the vanadyl cation. The stock solutions can be tested for partial oxidation by adding solid ascorbic acid (a reducing agent) and measuring any increase in the EPR intensity or in the absorbance at 750 nm due to formation of additional V02+ (25). The experiments using vanadyl cation were carried out in nitrogen-purged solutions and the pH was adjusted after the solutions were purged (20,25).
EXPERIMENTAL
Spectrophotometric determinations of initial rates of hydrolysis were obtained at 405 nm on a Lambda 4B Perkin-Elmer double-beam spectrophotometer equipped with a constant-temperature cell (25°C). The rates were determined by hydrolysis of p-nitrophenyl phosphate catalyzed by alkaline phosphatase at pH 8.00 (kO.05) and using a 100-s kinetic run measuring absorbances every 10 s. The addition of substrate or enzyme initiated the enzyme reaction in assay solutions that had been incubated for 2 min or more. The rates were determined in duplicate or triplicate. The inhibition of inorganic phosphate (side product) was kept to a minimum by monitoring the formation of p-nitrophenol to only about l-10% conversion of substrate. KC1 was added to the assay solution (1 M) in order to maintain a constant ionic strength. The rates were calculated using t = 18.5 liters mmol-’ cm-’ for p-nitrophenol anion and pro-
SECTION
Reagents, Enzymes, and Stock Solutions Chemicals were reagent grade (Fisher, Aldrich) and used without further purification. Water was distilled and deionized. Bovine intestinal mucosa alkaline phosphatase (EC 3.1.3.1) and di(cyclohexylammonium) p-nitrophenyl phosphate (PNPP) were purchased from Sigma and used without further purification. A standard 0.025 mM PNPP solution was prepared in doubly distilled water at neutral pH, divided into 2- to 4-ml aliquots, and frozen. A new frozen solution was used each day, and any remaining solution was discarded at the end of the day. The alkaline phosphatase was dissolved in doubly distilled water, and this stock solution was stored at 4°C. When 20% loss of activity (in V,,) was observed, a new stock solution was prepared. A vanadate
Kinetic Measurements
KINETIC
tein concentration Lowry (29).
was
quantified
METHOD
FOR
DETERMINATION
by the method
of
Alkaline Phosphatase Assay (VO,,f, Vi,+ V,, and Vi Determination for Vanadate in an Unknown Sample) Vo,ref. The VO,ref was measured in an assay solution containing 50 mM Hepes (or alternative buffer or concentration), 0.0256 mM p-nitrophenyl phosphate, 1 M KCl, complexing agent (5 mM Tricine), 5 X 10e6 M vanadate, the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. The Vi,ref was measured in an assay solution vi.ref. containing 50 mM Hepes (or alternative buffer or concentration), 0.0256 mM p-nitrophenyl phosphate, 1 M KCl, 5 X 10e6 M vanadate, the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. V,. The V0 was measured in an assay solution containing 50 mM Hepes (or alternative buffer in millimolar), 0.0256 mMp-nitrophenyl phosphate, 1 M KCl, complexing agent (5 mM Tricine), the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. Vi. The Vi was determined in an assay solution containing 50 mM Hepes (or alternative buffer or concentration), 0.0256 mM p-nitrophenyl phosphate, 1 M KCl, the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. The alkaline phosphatase assay (17,18,20) is very sensitive to contaminating trace metals. The buffers and HCl, HzS04, NaOH, and KC1 solutions for this work were therefore kept separated from common group supply. We have judged that a solution has become contaminated when the V, is significantly lower than the V, measured in the presence of 0.01 mM EDTA solution. Assays
Using Vanadyl Cation
Assays with vanadyl cation (20,25) were carried out as described above with minor modifications. The stock vanadyl cation solution contained 0.1 M H2S04. When significant concentrations of this solution were added to the assay, corresponding concentrations of 0.1 M NaOH were added to maintain the pH at 8.0. All solutions were purged with nitrogen before use to minimize oxidation by oxygen. The assay was carried out under nitrogen in a l-ml cuvette that was incubated for 2 min before the enzyme reaction was initiated. Vanadate is a weaker inhibitor than vanadyl cation, and oxidation of vanadyl cation will increase the measured reaction rates. Adequate purging of the solutions is a necessity: accurate determinations require purging for approximately 2 h. We normally check the reaction rates at 30-min intervals and only when the rate does not decrease further do we consider the solutions sufficiently purged. Vanadyl
OF
FREE
V(IV)
AND
cation can be accurately to lo-7 M. 51V NMR
-(V)
determined
55 in the range of 1O-8
Spectroscopy
Vanadium-51 is a sensitive, NMR-active nucleus of 99.75% natural abundance. Although its spin is 7/2, its linewidths are relatively narrow and easily resolved in the vanadium window. 51V NMR is therefore a convenient and informative tool for determination of vanadium(V) species in aqueous solutions (20,28,30,31). The 51V NMR spectra were recorded on a 200-MHz Bruker WPSY (4.7-T) spectrometer using an external lock. The accumulation parameters were a 16,000-Hz sweep width, a 90” pulse angle, an accumulation time of 0.2 s, and no relaxation delay. The chemical shifts are reported relative to the external reference standard, VOC& (0 ppm), although we in practice use an external reference solution (pH 8.8) containing the complex of vanadate and diethanolamine at -490 ppm (27,28). NMR
Sample Preparation
The vanadate solutions for 51V NMR studies were prepared by first mixing buffer and potassium chloride (1 M) (20,28). The pH and volume were adjusted to give the desired final values. The concentrations of vanadate oligomers determined in the presence of p-nitrophenyl phosphate and enzyme were within experimental error of those determined in the absence of p-nitrophenyl phosphate and enzyme. Substrate and enzyme were therefore, for experimental convenience, not included in the NMR studies. Data Manipulation The uncertainties in rate determinations ranged from 3 to 6%. In general we estimate that the uncertainties in the V,/Vi ratios (and in the inhibitor concentrations) will not exceed 12%, although high ionic strengths, multiple interfering species, and low concentrations of V(IV) may increase the experimental uncertainty to 16%. For example, we have observed deviations in V,/Vi ratios of up to 15% with different chemical lots of Hepes. These variations were reproducible and suggest that even reagent-grade chemicals contain small amounts of impurities that affect the activity of alkaline phosphatase (20). The kinetic data were analyzed using Cricket Graph, a program designed for statistical manipulations on the Apple computer. We estimate a 3 to 10% error in our rate constants and thus a 6 to 20% error in the V,/Vi ratio and inhibitor concentration. The high error limits occur at high ionic strengths and high concentrations of other substrates and inhibitors. The correlation coeflicients were 1.00 or 0.99 unless noted otherwise.
56
CRANS
RESULTS
Theoretical
AND
DISCUSSION
The reaction rate in an enzyme assay is dependent on the concentration of inhibitor(s) present in the assay solution. Rate changes can therefore be used to determine the concentration of an inhibitor. Assuming an inhibitor interacts with the enzyme by competitive inhibition, Michaelis-Menten kinetics can be used to describe a V,/ Vi ratio as a function of K, , V,,, , Ki , [S], and [I],
vo-
vnmxPI Km f [Sl Vma,[Sl . Krn(l + [IIIKJ + [Sl
V, represents the uninhibited rate and Vi the inhibited rate. Km and V,,, are Michaelis-Menten parameters for the enzyme reaction, Ki is the inhibition constant, [S] is the enzyme substrate concentration, and [I] is the concentration of inhibitor. Under conditions where substrate concentration, V,,, , Km, and Ki are constant, Eq. [l] simplifies (19,20) to
vo-
x-l+
AL.
be dependent only on [I] when stant. The relationship
Basis
vi-
ET
Km [II/K Km + tsl *
PI
+ Krn(l + [I,IIKl
[Ii], [12], and [I31 are con-
Km [II/K + [Izl/Kiz + [IJ/&)
+ [Sl
151
will therefore be valid if Vb is used as the uninhibited rate and Vi is the rate in the presence of I, Ii, 12, and 13. In addition to containing inhibitors, biological and environmental samples can contain compounds that are substrates for the enzyme. The enzyme substrate (A) used to monitor the enzyme reaction must compete with any additional substrate (B), and the rate, Vi, is expressed as
vg
=
F31
Note that the term Vmax,B[B]/KmB may not be observable at 405 mm ( vmax,B [B]/K,B g 0). Vy is the inhibited rate in the presence of I, A, and B and Vg is defined in Eq. [6] leading to the Vg/ Vl ratio as described by
The equation
[Ilref- m?E= ($-?)(K+F),
[3]
can be derived from Eq. [2] when Ki, Km, [S], and [I] are constant. Alkaline phosphatase is extremely sensitive to its environment and great care must be taken in performing the assays such that Ki and Km remain constant. The VO,ref/Vi,refratio is a Vo/Vi ratio determined in the presence of the sample plus a known concentration of inhibitor, and [Ileff is the effective concentration of inhibitor in the sample (20). Most biological and environmental samples contain many undefined components that could interfere with the enzyme assay reaction. If the sample contained three additional competitive inhibitors, Vi would need to be expressed as a function of four inhibitors (I, Ii, IB, 13),
vnmxts1
Vi = l+g+F+g+p 1
E41 +[S]
11
The inhibition caused by I,, counted for by measuring a rate taining all components except for est, I. The &/Vi ratio determined
13
IZ, and I3 can be acVb of the sample conthe inhibitor of interin this manner would
[71
If a Vg,,J Veref ratio is determined in which Vh,ref is the rate in the presence of all components in the sample solution except for I, and VZrefis the rate of the sample plus an additional known concentration of inhibitor, then Eq. [3] is modified to
Although biological samples or environmental samples are likely to contain both substrates and inhibitors, we show Eq. [3] and [8] to demonstrate that a linear relationship between the rate ratio and the inhibitor concentration should be observed. Standard addition methods should therefore in principal yield the proportionality factor q:
(2-2) =4 ([Ilref - bd. By measuring
the Vo,,J Vi,refratio at known
PI added vana-
KINETIC
METHOD
FOR
DETERMINATION
OF
FREE
V(W)
AND
57
-(V)
date concentration in the presence of the biological or environmental sample and the actual VO/Vi, a q factor can be calculated. Thus q is defined as the slope of the line in a plot of V,/Vi as a function of [I]. The unknown vanadate concentration in the sample, [I],e, can be calculated by
[IlefT=
$-1 i.
q. I/
[101
o0
5
10
15
20
Various Buffers
Determination of the appropriate q allows calculation of the vanadium concentration present in any sample. In practice it is not necessary to know the inhibitors and alternative substrates in the sample in order to determine q. The experimentally determined q in the presence of the sample will contain the appropriate components of [IldKi1, [I]dKiz, [AIIJLA, and [BIIKB. Using the standard addition technique (32) and measuring rates in the presence of a constant sample concentration, the appropriate q can be determined and thereby the vanadium concentration in the sample can be determined.
FIG. 1. The V,/V, ratios are shown for the inhibition of phosphate (0) and vanadate (fl) in the alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate in various buffers. The key to the buffers is as follows: 1 (Hepes), 2 (N-ethylmorpholine), 3 (Tris), 4 (BTP), 5 (EPPS), 6 (barbitol), 7 (TAPS), 8 (TES), 9 (PIPES), 10 (glycine), 11 (glycylglycine), 12 (pyridine), 13 (DEA), 14 (Bicine), 15 (MDEA), 16 (TEA), 17 (AMPSO), 18 (BES), 19 (DIPSO), 20 (IEA), 21 (TAPSO), and 22 (Tricine). The assays contained 0.025 mMp-nitrophenyl phosphate, 20 mM specified buffer, and 0.966 M KC1 at 23°C and pH 8.0 using 0.004 mg/ml alkaline phosphatase. The Vi was determined with 0.30 mMphosphate or 0.010 mMvanadate.
Selection of Reference Buffers Selection of Enzymes to Determine Vanadium Concentrations
Low-Level
In principle VO/Vi ratios can be used to determine a wide range of vanadium concentrations, given the appropriate enzyme reactions. This approach will be most sensitive when the substrate concentration is lower than twice K, and the Ki is of the same order of magnitude as the inhibitor concentration under determination. Therefore, the choice of enzyme system will limit the available observation range of inhibitor concentration. Vanadate and vanadyl cation inhibit ribonuclease, acid phosphatase, and alkaline phosphatase, with Ki’s in the concentration range 10e7 to 10e5 M (1,2). Acid and alkaline phosphatases have several simple and convenient enzyme assays, one of which involves the hydrolysis of p-nitrophenyl phosphate monitored by uv spectroscopy (20). The substrate specificity and conversion rate of both alkaline and acid phosphatases are very sensitive to the buffer and other compounds present in the assay solution. Biological samples are likely to contain several compounds that, due to the sensitivity of alkaline and acid phosphatase, will affect the rate of substrate hydrolysis. Application of the method described in this paper will be most successful if the assay solutions used for determining V,, Vi) VO,ref, and Vi,+ contain all the compounds present in the biological sample. Since the biological sample also contains the unknown concentration of vanadium, this must be removed in order to measure the I’,. We have found that adding a vanadium complexing agent to the assay solution at a concentration that does not affect the phosphatase allows sequestration of the vanadium in the sample.
All the enzyme reactions were conducted under standard assay conditions that involve a chosen reference buffer, pH, ionic strength, enzyme concentration, and temperature. The reference buffer is selected as a buffer in which the enzyme has a reasonable activity and in which little or no interaction occurs between the inhibitor and the buffer. Vanadate and vanadyl cation have the potential of interacting with hydroxyl and carboxylic acid groups in buffers. With the goal of selecting appropriate buffers, we have determined the Vo/Vi ratio for vanadate (0.010 mM) and phosphate (0.30 mM) in a series of 22 buffers and the results are illustrated in Fig. 1. Phosphate does not interact with the buffers and as a result a constant VO/Vi ratio is observed. Vanadate, on the other hand, interacts with several of the buffers and, as a result, the V,,/Vi ratios vary from 1 to 12. On the basis of the VO/Vi ratios we divide the buffers into three groups; the noninteracting or weakly interacting buffers (Vo/Vi > 6), the interacting buffers (5 > Vo/Vi > 2), and the strongly interacting buffers (V,/Vi < 2). Hepes, Nethylmorpholine, barbitol, and 2,4-lutidine appear to be the best buffers for vanadate studies in the neutral pH range, and Hepes and barbitol are the best buffers for vanadyl cation studies in the neutral pH range. Hepes was found to interact with vanadyl cation at high vanadyl concentrations; however, at low vanadyl concentrations this interaction was weak. We recommend working in barbitol (pK, 8.0), Hepes (pK, 7.6), N-ethylmorpholine (pK, 7.6), or 2,4-lutidine (pK, 7.0) when studies with both vanadyl cation and vanadate are to be carried out. Buffers such as triethanolamine (TEA), Bis-Tris propane (BTP), Tris, glycylglycine, Tricine, AMPSO, and
CRANS
b
ET
AL.
25
0.2 [v(Iv)],,,110-6
0.4
0.6 M
FIG. 2. (a) V,,/Vi ratios for the p-nitrophenyl phosphate hydrolysis catalyzed by alkaline phosphatase are plotted against total vanadate concentration (+), vanadate monomer concentration (m), vanadate dimer concentration (O), vanadate tetramer concentration (0), and vanadate pentamer concentration (A). The concentrations of various vanadate derivatives were determined using 51V NMR spectroscopy (+5% error limit) and the rates of hydrolysis were determined in 20 mM Hepes, 0.966 M KCl, 0.25 mM p-nitrophenyl phosphate at 23°C and pH 8.0 using from 0.007 to 0.04 mg/ml alkaline phosphatase. The correlation coefficient of the linear relationship between V,/Vi and monomeric vanadate was >0.995. (b) V,/V, ratios in various buffers are plotted against varying vanadate concentrations. The buffers are 50 mM Hepes ( X ), 50 mM Bis-Tris propane ( + ), 50 mM Hepes and 5 mM Tricine (A), 50 mM 2,4-lutidine (+), 50 mM N-ethylmorpholine (m), 50 mM barbitol, purged (0), 50 mM barbitol, unpurged (Cl). The correlation coefficients were all 0.99 or above. The enzyme rates were determined in the specified buffer, 1 M KCl, and 0.025 mMp-nitrophenylphosphate at 25°C and pH 8.0 using approximately 0.005 mg/ml alkaline phosphatase. (c) Vo/Vi ratios in various buffers are plotted against varying vanadyl cation concentrations. The buffers are as follows: 50 mM N-ethylmorpholine and 1 M KC1 ( X ), 50 mM N-ethylmorpholine and 0.5 M KC1 (0), 50 mM N-ethylmorpholine and 2.0 M KC1 (*), 50 mM barbitol (m), 50 mM Hepes (A), 50 mM Bis-Tris propane (A), and 50 mM 2,4-lutidine (0). The enzyme rates were determined in the specified buffers, in well-purged solutions, and with 1 M KC1 and 0.025 mMp-nitrophenyl phosphate at 25°C and pH 8.0 using approximately 0.005 mg/ml alkaline phosphatase.
DIPS0 should be avoided if both vanadium(IV) and (V) are to be measured. Most of these buffers form significant amounts of stable complexes with vanadate and/or vanadyl cation. Proportionality between V,lVi and Inhibitor Concentration The linear relationship between the V,/Vi ratio and the inhibitor concentration was examined experimentally for both vanadate and vanadyl cation (Figs. 2a-c). The inhibition by vanadate was first determined under conditions where the vanadate solutions consisted of complex mixtures of vanadate monomer, dimer, tetramer, and pentamer. The enzyme-catalyzed hydrolysis rates (Vi and VJ ofp-nitrophenyl phosphate (0.025 mM) were measured at 405 nm at vanadate concentrations ranging from 0 to 0.5 mM. The hydrolysis rates were measured with 0.007 to 0.04 mg/ml alkaline phosphatase in 20 mM Hepes and with 1 M KC1 (pH 8.0 at 25”C), and the V,/Vi ratios were calculated from these measurements. The concentrations of the various vanadate species in the assay solutions were also determined using 51VNMR spectroscopy. The details of these experiments are described under the Experimental Section. Plotting the V,/Vi ratio as a function of various vanadate derivatives shows a linear relationship with the vanadate monomer (R > 0.995) and nonlinear relationships with the vanadate dimer, tetramer, and pentamer. These re-
sults show the vanadate monomer is the only vanadate species in the solution that inhibits alkaline phosphatase significantly under the assay conditions. The inhibition by vanadate was also measured from 0 to 0.002 mM. Since the vanadate oligomerization reactions are in rapid equilibrium, the oligomers will always be present in solution; however, their concentrations will be very small. In the concentration range from 0 to 0.002 mM the vanadate monomer is for all practical purposes the only vanadate species present. The hydrolysis rates were measured in different buffers including Hepes, N-ethylmorpholine, 2,4-lutidine, barbitol, and BTP (Fig. 2b). The reaction rates were changed by purging the assay solution with nitrogen; however, these changes did not affect the V,/Vi ratio. Hepes has higher Vo/Vi ratios for vanadate than either N-ethylmorpoline, barbitol, BTP, or 2,4-lutidine, suggesting that these buffers interact more with vanadate than Hepes does. The inhibition by vanadyl cation was measured from 0 to 0.0007 mM vanadyl cation by monitoring the hydrolysis of p-nitrophenyl phosphate (0.025 mM). The reactions were carried out at several concentrations of barbito1 (20, 50, 67 mM) and in other buffers (50 mM Hepes, 50 mM 2,4-lutidine, 50 mM N-ethylmorpholine, and 50 mM BTP). Calculating the V,/V, ratios and plotting them as a function of the vanadyl concentration led to Fig. 2c. The curves in Fig. 2c show an initial linear region where the V,JVi ratio is proportional to the vanadyl cation concentration. As the V(W) concentration in-
KINETIC
. 0
METHOD
.
.
20
40
FOR
DETERMINATION
. 60
80
11 (Km + [S]) I lo3 w’ FIG. 3. The V,/lOV, ratio for vanadate (B) and the Vo/Vi ratio for vanadyl cation (Cl) are plotted as a function of l/(K,,, + [S]). The intercept on the Y-axis is l/lOK, or l/K,, respectively. The rate ratios for vanadate were determined in 20 mM Hepes, 0.966 M KCl, and 0.0025 to 0.50 mM p-nitrophenyl phosphate at 23°C at pH 8.0 using 0.0065 mg/ml alkaline phosphatase. The vanadate concentration was 0.010 mM. The correlation coefficient of the line was r0.995. The rate ratios for vanadyl cation were determined in 50 mM purged harhitol, 1 M KCl, and 0.0025 to 0.50 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase. The vanadyl cation concentration was 6.7 X 10-s M. The correlation coefficient of the line was 0.98.
creases, the concentration of free vanadyl cation as determined from the V,/Vi ratio levels off. Vanadyl cation is known to form oligomers or polymers at higher concentrations (25,26) and is therefore not expected to increase. Our observations are therefore consistent with previous reports suggesting that the maximum concentration of vanadyl cation is approximately 10m7M at neutral pH (25,26). Increasing the total vanadium concentration above 10m7 M will increase the overall vanadium(IV) concentration but will barely affect the free vanadyl cation concentration. Figure 2c also shows a noticeable decrease in the Vo/Vi ratio as the ionic strength increases. The proportionality factor q changes from 3.6 in 0.5 M KC1 to 2.5 in 1.0 M KC1 and to 1.5 at 2.0 M KCl. Consequently V,/Vi determinations at higher ionic strength will be more prone to error because of the smaller proportionality factor.
OF
FREE
V(W)
AND
59
-(V)
ear relationship as predicted in Eq. [2] is observed (R > 0.995). Corresponding experiments were conducted with 6.7 X lop8 M vanadyl cation using 50 InM barbitol as buffer. The V,/Vi ratios calculated from the observed rates are also plotted as a function of l/(K, + [S]) for vanadyl cation in Fig. 3. A linear relationship is also observed for this inhibitor (R = 0.98). We therefore conclude that the relationship shown in Eq. [2] is valid for both vanadate and vanadyl cation under the conditions examined in this work. Variations in the VO/V, Ratio as a Function of pH, Alkaline Phosphatase Concentration, and Temperature The changes in the V,/Vi ratios for vanadate, phosphate, and vanadyl cation were determined as a function of pH, enzyme concentration, and temperature. The hydrolysis rates ( Vi and V,) of p-nitrophenyl phosphate (0.025 mM) were measured from pH 7.8 to 8.5 with no inhibitor, with 0.010 mM vanadate, and with 0.30 mM phosphate. The hydrolysis rates were measured in 20 mM Hepes and 1 M KC1 (pH 8.0 at 25”C), and the V,/Vi ratios were calculated from these experiments. Corresponding experiments were carried out with vanadyl cation at 6.7 X 1O-8 in 50 mM barbitol. Plotting the V,/Vi ratios for vanadate, phosphate, and vanadyl cation as a function of pH shows that a considerable change in V,,/ Vi ratio takes place in the pH region under examination (Fig. 4). It is therefore important to maintain constant pH when determining V, and Vi, and we recommend keeping pH constant to within 0.05 pH unit. The variation of the Vo/Vi ratio as a function of enzyme concentration was determined with vanadate (0.010 mM), phosphate (0.30 mM), and vanadyl cation
+
-D V./IO”, (V(V))
“.I”1 ww *
(P,)
V./IO",
1.50 c. 2
1.25‘
co 1.00’
Dependence of the V,lVi Ratio on the Alkaline Phosphatase Substrate Concentration The linear relationship between the Vo/Vi ratio and the substrate (p-nitrophenyl phosphate) concentration was examined experimentally for vanadate and vanadyl cation. The hydrolysis rates (Vi and V,,) ofp-nitrophenyl phosphate (0.025 mM) were measured at substrate concentrations from 0.0050 to 0.50 mM in the presence of no inhibitor and in the presence of 0.010 mM vanadate. The hydrolysis rates were measured with 0.006 mg/ml alkaline phosphatase in 20 mM Hepes and with 1 M KC1 (pH 8.0 at 25”C), and the V,/Vi ratios were calculated from these measurements. The Vo/Vi ratios were plotted as a function of l/(K, + [S]) for vanadate in Fig. 3. The lin-
3
0.75
s
0.50
2
0.25’
i7
-
0,ooi
7.6
I 7.8
-
I 8.0
-
I 8.2
-
I 8.4
8.6
PH FIG. 4. The V,,/V, ratio for vanadyl cation (+) and the V,,/lOV, ratios for vanadate (0) and phosphate (w) are shown versus pH. The V,/lOV, ratios for 0.010 mM vanadate and 0.30 mM phosphate were determined in 20 mM Hepes, 0.966 M KCl, and 0.025 mMp-nitrophenyl phosphate at 23°C and pH 8.0 using 0.007 mg/ml alkaline phosphatase. The V,,/V, ratio for 6.7 X lo-* M vanadyl cation was determined in 50 mM purged barbitol, 1 M KCl, and 0.25 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase.
60
CRANS TABLE
V,/Vi Vanadyl tions
Ratios Cation
Determined at Various
1
for Vanadate, Phosphate, and Alkaline Phosphatase Concentra-
AL.
preincubation of the assay solution) when measuring VO, Vi 9Vo,ref7and Vi,refV,lVi Ratio Determination in a Solution Containing Multiple Components
VOlvi
[API
ET
X lo3 (mg/ml) 1.32 1.8 2.2 2.6 2.7 3.1 3.5 3.8 4.2 4.4 5.2 6.2
Average Standard deviation 95% confidence limit a The V, was determined in mM p-nitrophenyl phosphate mined with 0.010 mM vanadate. b The V, was determined in mM p-nitrophenyl phosphate mined with 0.30 mM phosphate. ’ The V, was determined in mM p-nitrophenyl phosphate mined with 6.7 X 10-s M vanadyl
VW)
a
Pb
11.4 -
2.86
11.7 10.8
3.00 2.95
11.6 11.8
2.94 2.99
11.4 0.40 0.19
2.95 0.055 0.026
V(IV)C 1.16 1.16 1.13 1.20 1.18 1.14 1.11
1.15 0.030 0.040
20 mM Hepes, 0.966 M KCl, and 0.025 at pH 8.0 and 23°C. The Vi was deter20 mM Hepes, 0.966 M KCI, and 0.025 at pH 8.0 and 23°C. The Vi was deter50 mM barbitol, 1.0 M KCl, and 0.025 at pH 8.0 and 25°C. The Vi was detercation.
(6.7 X lo-’ M) by measuring the hydrolysis rate of p-nitrophenyl phosphate (0.025 mM). The vanadate and phosphate measurements were conducted in 20 mM Hepes and the vanadyl cation experiments in 50 mM barbitol. The enzyme concentration varied from 0.002 to 0.007 mg/ml, and the resulting rates were used to calculate the Vo/Vi ratios. The results are given in Table 1. Changing the enzyme concentration caused no apparent change in the V,/Vi ratio. The concentration of alkaline phosphatase used in the assays should therefore not affect the Vo/Vi ratio as long as both V, and Vi are determined accurately. The variation of the V,/Vi ratio as a function of temperature was determined for vanadate at 0.002 and 0.010 mM (Table 2). The V,,/ Vi ratio changed from 2.85 at 20°C to 2.42 at 35°C for 0.002 mM vanadate. At 0.010 mM vanadate the Vo/Vi ratio changed from 9.30 at 20°C to 7.60 at 35°C (Table 2). Temperature changes are therefore likely to affect the Vo/Vi ratio significantly. Even a 1-2” change caused by adding a cool substrate solution into an incubated assay solution could affect the V,/Vi ratio by 5510%. We therefore recommend that care be taken in carrying out the enzyme assay using identical experimental technique (preferably including 2 min or longer
The linear relationship between V,lVi and vanadate monomer concentration should not be affected by additional substrates and inhibitors in the assay mixture (as predicted by Eqs. [6]-[8]). Such relationships were examined by measuring the rates of hydrolysis at varying concentrations of vanadate in the presence of 0.025 mM p-nitrophenyl phosphate and an additional inhibitor (0.5 mM phosphate) or enzyme substrate (0.5 mM glycerol 3-phosphate). The V,/ Vi ratios were calculated from the rates and plotted as a function of vanadate concentration and are shown in Fig. 5. Linear relationships between the V,/Vi ratio and the vanadate concentration are observed in all cases although the presence of additional inhibitors and/or substrates in the assay solutions decreased the proportionality factor, q. Application of the Alkaline Phosphatase Assay In biological systems vanadium readily converts from oxidation state V to IV and vice versa. Since the oxidation state of vanadium is crucial to how vanadium acts, it is of interest to determine the concentrations of both free oxidation states IV and V. In this section we demonstrate how either V(N) and V(V) can be determined in solutions containing either or both V(IV) and V(V). The Vo/Vi ratio expresses the concentration of both free V(V) and V(IV), if the V, is determined in the presence of the sample and a complexing agent of both vanadium(N) and (V) (such as DIPS0 or Tricine) (20). All components in the sample will affect the V,, in a manner similar to Vi, and thus only the concentration of V(V)
TABLE
Vo/Vi Ratios Vanadate
Determined at Various
2 for 2 X lo-’ and Temperatures”
10e5 M
VolK Temperature (“0
20 22.5 25 27.5 30 32.5 35
2 x 10-6M
1o-5
VW
VW)
2.85 2.79 2.64 2.48 2.46 2.48 2.42
a The V, was determined in 50 mM Hepes, 1 M KCl, p-nitrophenyl phosphate at pH 8.0 using approximately alkaline phosphatase.
M
9.30 8.78 8.77 8.19 8.14 7.88 7.60 and 0.025 mM 0.005 mg/ml
KINETIC
5 ‘0 >
METHOD
FOR
DETERMINATION
15.
1.0
0.5 0.0
1.0
[V(V)]
2.0
11O-6 M
FIG. 5. The V,/V, ratios in the presence of various additional inhihitors, substrates and complexing agents are shown for vanadate. The additional compounds are: none ( + ), 2.5 mM BTP (*), 10m7 M vanadyl cation (+), 0.5 mM glycerol 3-phosphate (W), 0.5 mM phosphate (O), 0.5 mM glycerol 3-phosphate and 0.5 mM phosphate (A), 2.5 mM BTP and 10m7 M vanadyl cation ( X ), and 5 mM Tricine (A). The V,/V, ratios were determined using the above components, 50 mM Hepes, 1 M KCI, and 0.025 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase. The correlation coefficients were 0.99 or above unless specified otherwise.
and V(IV) will dictate the V,/Vi ratio according to Eq. [8]-[lo]. Similarly, the V,/Vi ratio could be used to measure the concentration of free V(W), if V, is determined in the presence of the sample and a V(W)-specific complexing agent such as BTP. The Vo/Vi ratio could also be used to measure the concentration of V(V) if V,, is determined in the presence of a complexing agent specific for V(V). This approach was demonstrated by measuring the V,/ Vi ratios for a solution containing a mixture of vanadate and vanadyl cation. The rates of hydrolysis ( V, and Vi) of p-nitrophenyl phosphate were measured at varying vanadate concentrations in the presence of 1.3 X 10e7 M vanadyl cation (approximately 35% inhibition) and a linear relationship was observed when plotting the V,/ Vi as a function of [V(V)] (Fig. 5). The rates obtained in the presence of Tricine, vanadyl cation, and various concentrations of vanadate should therefore be identical with a V,, obtained in the absence of either vanadate or vanadyl cation, and this was indeed observed (Fig. 5). A corresponding series of rate measurements carried out in the presence of 1.3 X 10e7M vanadyl cation, BTP, and varying vanadate concentrations again yielded a linear relationship with a nonzero proportionality factor q. This is in accord with the expectation that BTP selectively complexes vanadyl cation. By combining the measurements using both BTP and Tricine, the V(IV) concentration, the total vanadium concentration, and the V(V) concentration can be calculated. The Vo/Vi ratio was determined in barbitol in the presence of 0.001 mM vanadate with varying concentra-
OF
FREE
V(IV)
AND
-(V)
61
tions of vanadyl cation. A linear relationship was observed between the V,/Vi ratio and the [VO”‘] at low concentrations and is shown in Fig. 6. The rates obtained in the presence of BTP and varying vanadyl concentrations should be identical with the rate obtained without vanadyl cation but with 0.001 mM vanadate. This was indeed observed. The Vo/Vi ratios determined in the presence of 5 mM Tricine were 1.0 in accord with the expectation that Tricine would complex this range of vanadyl cation concentrations. An attempt to determine the free vanadyl cation concentration in the presence of 0.5 mM phosphate was complicated by the fact that phosphate complexed the vanadyl cation. As a result very little (less than 0.03 X lo-’ M) of the original (from 0.03 to 0.7 X lop8 M) vanadyl cation was free in these assay solutions containing phosphate. Glycerol 3-phosphate (0.5 mM) was also found to complex the vanadyl cation but much less than phosphate. The proportionality factor q in a plot of V,/Vi as a function of vanadyl cation concentration was >l (fO.l) in the presence of 0.5 mM glycerol 3-phosphate, suggesting that not all the vanadyl cation was complexed by glycerol 3-phosphate. The variation in vanadyl cation concentration in the presence of glycerol 3-phosphate can be determined using the assay. These experiments illustrate the ability of many biomolecules to complex vanadyl cation. Complex fluids with high concentrations of vanadyl-complexing compounds are therefore likely to contain both the complexed vanadyl cation and only little free vanadyl cation (25,26). In order to determine the concentration of free vanadyl cation in such a biological sample containing complexing agents, such agents must be present when measuring the proportionality factor.
0.84
0.0
-
m
0.2 [V(lV)]tot
0.4
0.6
/l O-6 M
FIG. 6. The V,,/Vi ratio in the presence of various additional inhibitors, substrates and complexing agents are shown for vanadyl cation. The additional compounds are: none (A), 2.5 mM BTP (O), 10m6 M vanadate ( + ), 0.5 mM glycerol 3-phosphate (m), 0.5 mM phosphate (+), 10m5 M vanadate and 2.5 mM BTP (O), and 10m6 M vanadate and 5 mM Tricine ( X ). The Vo/Vi ratios were determined using the above components, 50 mM barbitol, 1 M KCl, and 0.025 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase.
62
CRANS
The described method is applicable when Km, V,,,,,, are constant under the various rate determinations. Such assumptions are nontrivial with alkaline phosphatase, because alkaline phosphatase has Michaelis-Menten constants that are extremely sensitive to the environment. Variations in buffer, ionic strength, temperature, and pH have been shown to change the Km and Ki for vanadate by several orders of magnitude (17,X3). Using the V,/Vi ratio to determine [IleR, one can circumvent the problem of the extreme sensitivity to environment by using the standard addition technique (32). The experimental determination of V, and VO,ref will require a complexing agent that will specifically bind the species of interest. We have used BTP to selectively complex vanadium(N) because this buffer will complex vanadium(rV) in the concentration range 10-s to 10e6 M without binding vanadium(V) in the concentration range 10e7 to 10e5 M. Other sequestering agents could substitute for BTP if this buffer causes problems in any particular samples. If a substitute for BTP is needed, a potential candidate should not change the V, of a reference buffer more than 1620%. This requirement is based on the observations that the rates of the reaction catalyzed by alkaline phosphatase are related to the selectivity with which alkaline phosphatase binds substrate and/or inhibitor. If no changes in the reaction rates are observed in the presence of the ligand, the ligand does not appear to affect the Vo/Vi ratio. Quantitative determination of vanadium concentrations can therefore be obtained by measuring Vo, Vi, VO,ref, and Vi,refp where V, and VO,re.are determined in the presence of the specific sequestering agent, Vi and Vi,ref are determined without such a compound, and a known concentration of vanadium is added
Ki, and KJK,
to
Vi,ref*
The sensitivity of alkaline phosphatase to its environment may render this assay inappropriate for quantitative determinations with some samples. The following control experiment will demonstrate whether such conditions indeed prevail. The experiment involves the determination of the V,/Vi ratio for an innocuous (to the sample) competitive inhibitor that is known not to be present in the sample under investigation. The corresponding V,/Vi ratio of this inhibitor in the presence of the biological environmental sample is also determined. Examples of such inhibitors are phosphate, molybdate, arsenate, and tungstate. If the two V,/Vi ratios are identical, the assay conditions are compatible with the biological sample. On the other hand, if the V,/Vi in the presence of biological or environmental sample is significantly larger or smaller (> or
188,53-64
(1990)
A Kinetic Method for Determination of Free Vanadium(W) and (V) at Trace Level Concentrations D. C. Crans,l
M. Shaia Gottlieb,
Department
of Chemistry,
Received
17,1989
July
Colorado
J. Tawara,
State University,
R. L. Bunch,
A kinetic method based on alkaline phosphatase has been developed to measure free trace levels of vanadium(IV) and (V). The method involves measuring the rate of the alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate with (VJ and without (V,) a competitive inhibitor in the assay. Michaelis-Menten kinetics for a competitive inhibitor was used to express the relationship between Vo/Vi and the inhibitor concentration. Measuring both V,-, and Vi thus yields a Vo/ Vi ratio that allows calculation of the competitive inhibitor concentration. Determination of free vanadium in complex fluids can be accomplished by comparing the ratio of rates of p-nitrophenyl phosphate hydrolysis with and without a sequestering agent to the ratios of rates measured on addition of a known vanadium concentration. Free vanadium(V) can conveniently be measured from lo-’ to lo-’ M and free vanadium(IV) can be measured at 10-s M and above. The error limits on the vanadium determinations range from +3 to +-12% of the concentration under investigation depending on the conditions under which the assay was conducted. 0 1990 Academic Press. Inc.
Vanadium is an important dietary trace element (1,2). The major sources of vanadium intake in mammals come from seeds,grains, roots, and water. Vanadium has a number of biological effects, including normalizing glucose levels and stimulation of hexose transport. It is an insulin mimetic agent, and it affects the cardiac muscle in mammals (1,2). It is a potent inhibitor for ribonucleases, ATPases, nucleases, and phosphatases and is a required cofactor in some nitrogenases (3,4) and bromoperoxidases (5-8). Organic vanadate derivatives can be substrates for glycolytic and pentose phosphate shunt enzymes (9-11). Vanadium(IV) is the major form pres1 To whom 0003.2697/90 Copyright All rights
correspondence
should
$3.00 0 1990 by Academic Press, of reproduction in any form
be addressed.
and L. A. Theisen
Fort Collins, Colorado
80523
ent in mammals. Tunicates have also been found to contain vanadium(II1) (12). Available analytical techniques can measure total vanadium or vanadium in oxidation states III, IV, or V (13). Oxidation states IV and V interconvert readily under physiological conditions, and it is often a problem to determine the active vanadium species in biological studies (1,2). Consequently, we present an enzymatic method that will measure free vanadium(IV) and (V) simultaneously in the concentration range from 10m5to lo-’ M vanadium. Analytical methods currently used to determine trace levels of vanadium include atomic absorption spectrometry (AAS’; detection range, parts per billion), neutron activation analysis (NAA; detection limit, 10-l’ M), inductively coupled plasma (ICP; detection range, from 10e3 to 10e6 g/g), dc plasma atomic emission spectrophotometry (DCPAES; detection range, from lop6 to 3 X lo-’ g/ml), mass spectrometry (detection range, parts per million), electron paramagnetic resonance spectroscopy (EPR; detection range, from lop5 to 1O-4 M), and nuclear magnetic resonance (NMR; detection limit, 1O-5 M) (12). With the exception of EPR and NMR spectroscopy, the above methods measure total vanadium con-
’ Abbreviations: AAS, atomic absorption spectrometry; NAA, neutron activation analysis; ICP, inductively coupled plasma; DCPAES, dc plasma atomic emission spectrophotometry; EPR, electron paramagnetic resonance spectroscopy; BTP or Bis-Tris propane, 1,3-bis[(tris(hydroxymethyl)methyl)amino]propane; EPPS, iV’-(2hydroxyethyl)-N-piperazinepropanesulfonic acid; TAPS, 3-[(tris(hydroxymethyl)methyl)amino]propanesulfonic acid, TES, 2[(tris(hydroxymethyl)methyl)amino]ethanesulfonic acid; PIPES, piperazine-N,N’-b&(2-ethanesulfonic acid); DEA, diethanolamine; Bicine, N,N-bis(2-hydroxyethyl)glycine; NEM, N-ethylmorpholine, MDEA, N-methyldiethanolamine; TEA, triethanolamine; AMPSO, 2-[(l,l-dimethyl-2-hydroxyethyl)amino]ethanesulfonic acid; DIPSO, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid; IEA, triisopropanolamine; TAPSO, 3-[N-(tris(hydroxymethyl)methyl)amino]-2-hydroxypropanesulfonic acid; Tricine, N-[tris(hydroxymethyl)methyl]glycine; Bis-Tris, [bis(2-hydroxyethyl)amino]tris[(hydroxymethyl)methane]; MIDA, N-methyliminodiacetic acid; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid. 53
Inc. reserved.
54
CRANS
ET
AL.
centrations and do not distinguish between different vanadium species or oxidation states. EPR will detect only V(IV) species and 51V NMR will detect only V(V) species. Studies requiring determination of both V(IV) and V(V) concentrations therefore involve experiments using both EPR and 51V NMR. Although such studies have been conducted in the past, the low sensitivity, the nonroutine instrumental requirements of both EPR and NMR, and the nonroutine use of EPR make such studies difficult to coordinate and quantitate (1,2,13). Biological studies of vanadium would benefit from an experimentally simple quantitative technique. Kinetic methods have previously been used to determine trace level organic compounds, inorganic compounds, and proteins in biological fluids (14-16). We present a kinetic method based on the alkaline phosphatase catalyzed hydrolysis of p-nitrophenyl phosphate to form p-nitrophenol and phosphate. Alkaline phosphatase hydrolyzes p-nitrophenyl phosphate with an uninhibited rate, VO; however, in the presence of a competitive inhibitor, the inhibited rate, Vi, will reflect the concentration of inhibitor. Using Michaelis-Menten kinetics, a direct relationship can be derived between the competitive inhibitor concentration [IleE and the V,,/ Vi ratio (17-20). Alkaline phosphatase is competitively inhibited by phosphate and phosphate analogs including arsenate, vanadate, and molybdate (17,18,21-24). Vanadate is a much more potent inhibitor (Ki - 10e6 M) than phosphate (Ki - 10e3 M), possibly because a trigonal-bipyramidal vanadate-enzyme complex (transition state analog) is formed (1,21-24). Vanadyl cation is also a potent competitive inhibitor for alkaline phosphatase. The hydrolysis rates of p-nitrophenyl phosphate are therefore a sensitive measure for low concentrations of vanadate and vanadyl cation. This approach has been developed to measure concentrations of free vanadate and/or vanadyl cation (simultaneously) at lo-’ to 1O-5 M levels in complex samples.
stock solution was prepared by dissolving appropriately weighed vanadium pentoxide (V,OJ with 2 eq of sodium hydroxide to generate a vanadate solution of approximately 0.25 M; this solution was stored at 4°C. The vanadyl cation stock solution was prepared by dissolving appropriately weighed vanadyl sulfate (VOS04.H20) into 0.1 M H2S04 to generate a vanadyl sulfate solution of approximately 0.5 M (25). No changes in the concentrations of the vanadate or vanadyl cation standard solutions, which were monitored over the course of 6 months, were seen. The exact concentration of the vanadate stock solution was determined by diluting the solution lOOO-fold into 0.1 M NaOH and measuring the absorbance at 260 nm. The major (“only”) vanadate species in this solution is HVOi- , and by using an extinction coefficient of 3.55 X lo3 M-l cm-’ the concentration of the diluted stock and original stock could be calculated (27). At no time was acid added to a solution containing vanadate, since vanadate in the presence of acid generates the orange decamer (1,2,28). The exact concentration of the vanadyl stock solution was determined by measuring the absorbance of this solution at 750 nm. The concentration was then calculated using a molar extinction coefficient of E= 18.0 -t 0.2 M-l cm-’ (25). Alternatively, the concentration of the vanadyl cation stock solution could be determined using a permanganate-oxalate titration procedure (26). The stock solutions were kept at 4°C. Since the pH < 2, we observed no oxidation of the vanadyl cation. The stock solutions can be tested for partial oxidation by adding solid ascorbic acid (a reducing agent) and measuring any increase in the EPR intensity or in the absorbance at 750 nm due to formation of additional V02+ (25). The experiments using vanadyl cation were carried out in nitrogen-purged solutions and the pH was adjusted after the solutions were purged (20,25).
EXPERIMENTAL
Spectrophotometric determinations of initial rates of hydrolysis were obtained at 405 nm on a Lambda 4B Perkin-Elmer double-beam spectrophotometer equipped with a constant-temperature cell (25°C). The rates were determined by hydrolysis of p-nitrophenyl phosphate catalyzed by alkaline phosphatase at pH 8.00 (kO.05) and using a 100-s kinetic run measuring absorbances every 10 s. The addition of substrate or enzyme initiated the enzyme reaction in assay solutions that had been incubated for 2 min or more. The rates were determined in duplicate or triplicate. The inhibition of inorganic phosphate (side product) was kept to a minimum by monitoring the formation of p-nitrophenol to only about l-10% conversion of substrate. KC1 was added to the assay solution (1 M) in order to maintain a constant ionic strength. The rates were calculated using t = 18.5 liters mmol-’ cm-’ for p-nitrophenol anion and pro-
SECTION
Reagents, Enzymes, and Stock Solutions Chemicals were reagent grade (Fisher, Aldrich) and used without further purification. Water was distilled and deionized. Bovine intestinal mucosa alkaline phosphatase (EC 3.1.3.1) and di(cyclohexylammonium) p-nitrophenyl phosphate (PNPP) were purchased from Sigma and used without further purification. A standard 0.025 mM PNPP solution was prepared in doubly distilled water at neutral pH, divided into 2- to 4-ml aliquots, and frozen. A new frozen solution was used each day, and any remaining solution was discarded at the end of the day. The alkaline phosphatase was dissolved in doubly distilled water, and this stock solution was stored at 4°C. When 20% loss of activity (in V,,) was observed, a new stock solution was prepared. A vanadate
Kinetic Measurements
KINETIC
tein concentration Lowry (29).
was
quantified
METHOD
FOR
DETERMINATION
by the method
of
Alkaline Phosphatase Assay (VO,,f, Vi,+ V,, and Vi Determination for Vanadate in an Unknown Sample) Vo,ref. The VO,ref was measured in an assay solution containing 50 mM Hepes (or alternative buffer or concentration), 0.0256 mM p-nitrophenyl phosphate, 1 M KCl, complexing agent (5 mM Tricine), 5 X 10e6 M vanadate, the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. The Vi,ref was measured in an assay solution vi.ref. containing 50 mM Hepes (or alternative buffer or concentration), 0.0256 mM p-nitrophenyl phosphate, 1 M KCl, 5 X 10e6 M vanadate, the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. V,. The V0 was measured in an assay solution containing 50 mM Hepes (or alternative buffer in millimolar), 0.0256 mMp-nitrophenyl phosphate, 1 M KCl, complexing agent (5 mM Tricine), the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. Vi. The Vi was determined in an assay solution containing 50 mM Hepes (or alternative buffer or concentration), 0.0256 mM p-nitrophenyl phosphate, 1 M KCl, the sample under examination, and 0.003 to 0.007 mg alkaline phosphatase. The alkaline phosphatase assay (17,18,20) is very sensitive to contaminating trace metals. The buffers and HCl, HzS04, NaOH, and KC1 solutions for this work were therefore kept separated from common group supply. We have judged that a solution has become contaminated when the V, is significantly lower than the V, measured in the presence of 0.01 mM EDTA solution. Assays
Using Vanadyl Cation
Assays with vanadyl cation (20,25) were carried out as described above with minor modifications. The stock vanadyl cation solution contained 0.1 M H2S04. When significant concentrations of this solution were added to the assay, corresponding concentrations of 0.1 M NaOH were added to maintain the pH at 8.0. All solutions were purged with nitrogen before use to minimize oxidation by oxygen. The assay was carried out under nitrogen in a l-ml cuvette that was incubated for 2 min before the enzyme reaction was initiated. Vanadate is a weaker inhibitor than vanadyl cation, and oxidation of vanadyl cation will increase the measured reaction rates. Adequate purging of the solutions is a necessity: accurate determinations require purging for approximately 2 h. We normally check the reaction rates at 30-min intervals and only when the rate does not decrease further do we consider the solutions sufficiently purged. Vanadyl
OF
FREE
V(IV)
AND
cation can be accurately to lo-7 M. 51V NMR
-(V)
determined
55 in the range of 1O-8
Spectroscopy
Vanadium-51 is a sensitive, NMR-active nucleus of 99.75% natural abundance. Although its spin is 7/2, its linewidths are relatively narrow and easily resolved in the vanadium window. 51V NMR is therefore a convenient and informative tool for determination of vanadium(V) species in aqueous solutions (20,28,30,31). The 51V NMR spectra were recorded on a 200-MHz Bruker WPSY (4.7-T) spectrometer using an external lock. The accumulation parameters were a 16,000-Hz sweep width, a 90” pulse angle, an accumulation time of 0.2 s, and no relaxation delay. The chemical shifts are reported relative to the external reference standard, VOC& (0 ppm), although we in practice use an external reference solution (pH 8.8) containing the complex of vanadate and diethanolamine at -490 ppm (27,28). NMR
Sample Preparation
The vanadate solutions for 51V NMR studies were prepared by first mixing buffer and potassium chloride (1 M) (20,28). The pH and volume were adjusted to give the desired final values. The concentrations of vanadate oligomers determined in the presence of p-nitrophenyl phosphate and enzyme were within experimental error of those determined in the absence of p-nitrophenyl phosphate and enzyme. Substrate and enzyme were therefore, for experimental convenience, not included in the NMR studies. Data Manipulation The uncertainties in rate determinations ranged from 3 to 6%. In general we estimate that the uncertainties in the V,/Vi ratios (and in the inhibitor concentrations) will not exceed 12%, although high ionic strengths, multiple interfering species, and low concentrations of V(IV) may increase the experimental uncertainty to 16%. For example, we have observed deviations in V,/Vi ratios of up to 15% with different chemical lots of Hepes. These variations were reproducible and suggest that even reagent-grade chemicals contain small amounts of impurities that affect the activity of alkaline phosphatase (20). The kinetic data were analyzed using Cricket Graph, a program designed for statistical manipulations on the Apple computer. We estimate a 3 to 10% error in our rate constants and thus a 6 to 20% error in the V,/Vi ratio and inhibitor concentration. The high error limits occur at high ionic strengths and high concentrations of other substrates and inhibitors. The correlation coeflicients were 1.00 or 0.99 unless noted otherwise.
56
CRANS
RESULTS
Theoretical
AND
DISCUSSION
The reaction rate in an enzyme assay is dependent on the concentration of inhibitor(s) present in the assay solution. Rate changes can therefore be used to determine the concentration of an inhibitor. Assuming an inhibitor interacts with the enzyme by competitive inhibition, Michaelis-Menten kinetics can be used to describe a V,/ Vi ratio as a function of K, , V,,, , Ki , [S], and [I],
vo-
vnmxPI Km f [Sl Vma,[Sl . Krn(l + [IIIKJ + [Sl
V, represents the uninhibited rate and Vi the inhibited rate. Km and V,,, are Michaelis-Menten parameters for the enzyme reaction, Ki is the inhibition constant, [S] is the enzyme substrate concentration, and [I] is the concentration of inhibitor. Under conditions where substrate concentration, V,,, , Km, and Ki are constant, Eq. [l] simplifies (19,20) to
vo-
x-l+
AL.
be dependent only on [I] when stant. The relationship
Basis
vi-
ET
Km [II/K Km + tsl *
PI
+ Krn(l + [I,IIKl
[Ii], [12], and [I31 are con-
Km [II/K + [Izl/Kiz + [IJ/&)
+ [Sl
151
will therefore be valid if Vb is used as the uninhibited rate and Vi is the rate in the presence of I, Ii, 12, and 13. In addition to containing inhibitors, biological and environmental samples can contain compounds that are substrates for the enzyme. The enzyme substrate (A) used to monitor the enzyme reaction must compete with any additional substrate (B), and the rate, Vi, is expressed as
vg
=
F31
Note that the term Vmax,B[B]/KmB may not be observable at 405 mm ( vmax,B [B]/K,B g 0). Vy is the inhibited rate in the presence of I, A, and B and Vg is defined in Eq. [6] leading to the Vg/ Vl ratio as described by
The equation
[Ilref- m?E= ($-?)(K+F),
[3]
can be derived from Eq. [2] when Ki, Km, [S], and [I] are constant. Alkaline phosphatase is extremely sensitive to its environment and great care must be taken in performing the assays such that Ki and Km remain constant. The VO,ref/Vi,refratio is a Vo/Vi ratio determined in the presence of the sample plus a known concentration of inhibitor, and [Ileff is the effective concentration of inhibitor in the sample (20). Most biological and environmental samples contain many undefined components that could interfere with the enzyme assay reaction. If the sample contained three additional competitive inhibitors, Vi would need to be expressed as a function of four inhibitors (I, Ii, IB, 13),
vnmxts1
Vi = l+g+F+g+p 1
E41 +[S]
11
The inhibition caused by I,, counted for by measuring a rate taining all components except for est, I. The &/Vi ratio determined
13
IZ, and I3 can be acVb of the sample conthe inhibitor of interin this manner would
[71
If a Vg,,J Veref ratio is determined in which Vh,ref is the rate in the presence of all components in the sample solution except for I, and VZrefis the rate of the sample plus an additional known concentration of inhibitor, then Eq. [3] is modified to
Although biological samples or environmental samples are likely to contain both substrates and inhibitors, we show Eq. [3] and [8] to demonstrate that a linear relationship between the rate ratio and the inhibitor concentration should be observed. Standard addition methods should therefore in principal yield the proportionality factor q:
(2-2) =4 ([Ilref - bd. By measuring
the Vo,,J Vi,refratio at known
PI added vana-
KINETIC
METHOD
FOR
DETERMINATION
OF
FREE
V(W)
AND
57
-(V)
date concentration in the presence of the biological or environmental sample and the actual VO/Vi, a q factor can be calculated. Thus q is defined as the slope of the line in a plot of V,/Vi as a function of [I]. The unknown vanadate concentration in the sample, [I],e, can be calculated by
[IlefT=
$-1 i.
q. I/
[101
o0
5
10
15
20
Various Buffers
Determination of the appropriate q allows calculation of the vanadium concentration present in any sample. In practice it is not necessary to know the inhibitors and alternative substrates in the sample in order to determine q. The experimentally determined q in the presence of the sample will contain the appropriate components of [IldKi1, [I]dKiz, [AIIJLA, and [BIIKB. Using the standard addition technique (32) and measuring rates in the presence of a constant sample concentration, the appropriate q can be determined and thereby the vanadium concentration in the sample can be determined.
FIG. 1. The V,/V, ratios are shown for the inhibition of phosphate (0) and vanadate (fl) in the alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate in various buffers. The key to the buffers is as follows: 1 (Hepes), 2 (N-ethylmorpholine), 3 (Tris), 4 (BTP), 5 (EPPS), 6 (barbitol), 7 (TAPS), 8 (TES), 9 (PIPES), 10 (glycine), 11 (glycylglycine), 12 (pyridine), 13 (DEA), 14 (Bicine), 15 (MDEA), 16 (TEA), 17 (AMPSO), 18 (BES), 19 (DIPSO), 20 (IEA), 21 (TAPSO), and 22 (Tricine). The assays contained 0.025 mMp-nitrophenyl phosphate, 20 mM specified buffer, and 0.966 M KC1 at 23°C and pH 8.0 using 0.004 mg/ml alkaline phosphatase. The Vi was determined with 0.30 mMphosphate or 0.010 mMvanadate.
Selection of Reference Buffers Selection of Enzymes to Determine Vanadium Concentrations
Low-Level
In principle VO/Vi ratios can be used to determine a wide range of vanadium concentrations, given the appropriate enzyme reactions. This approach will be most sensitive when the substrate concentration is lower than twice K, and the Ki is of the same order of magnitude as the inhibitor concentration under determination. Therefore, the choice of enzyme system will limit the available observation range of inhibitor concentration. Vanadate and vanadyl cation inhibit ribonuclease, acid phosphatase, and alkaline phosphatase, with Ki’s in the concentration range 10e7 to 10e5 M (1,2). Acid and alkaline phosphatases have several simple and convenient enzyme assays, one of which involves the hydrolysis of p-nitrophenyl phosphate monitored by uv spectroscopy (20). The substrate specificity and conversion rate of both alkaline and acid phosphatases are very sensitive to the buffer and other compounds present in the assay solution. Biological samples are likely to contain several compounds that, due to the sensitivity of alkaline and acid phosphatase, will affect the rate of substrate hydrolysis. Application of the method described in this paper will be most successful if the assay solutions used for determining V,, Vi) VO,ref, and Vi,+ contain all the compounds present in the biological sample. Since the biological sample also contains the unknown concentration of vanadium, this must be removed in order to measure the I’,. We have found that adding a vanadium complexing agent to the assay solution at a concentration that does not affect the phosphatase allows sequestration of the vanadium in the sample.
All the enzyme reactions were conducted under standard assay conditions that involve a chosen reference buffer, pH, ionic strength, enzyme concentration, and temperature. The reference buffer is selected as a buffer in which the enzyme has a reasonable activity and in which little or no interaction occurs between the inhibitor and the buffer. Vanadate and vanadyl cation have the potential of interacting with hydroxyl and carboxylic acid groups in buffers. With the goal of selecting appropriate buffers, we have determined the Vo/Vi ratio for vanadate (0.010 mM) and phosphate (0.30 mM) in a series of 22 buffers and the results are illustrated in Fig. 1. Phosphate does not interact with the buffers and as a result a constant VO/Vi ratio is observed. Vanadate, on the other hand, interacts with several of the buffers and, as a result, the V,,/Vi ratios vary from 1 to 12. On the basis of the VO/Vi ratios we divide the buffers into three groups; the noninteracting or weakly interacting buffers (Vo/Vi > 6), the interacting buffers (5 > Vo/Vi > 2), and the strongly interacting buffers (V,/Vi < 2). Hepes, Nethylmorpholine, barbitol, and 2,4-lutidine appear to be the best buffers for vanadate studies in the neutral pH range, and Hepes and barbitol are the best buffers for vanadyl cation studies in the neutral pH range. Hepes was found to interact with vanadyl cation at high vanadyl concentrations; however, at low vanadyl concentrations this interaction was weak. We recommend working in barbitol (pK, 8.0), Hepes (pK, 7.6), N-ethylmorpholine (pK, 7.6), or 2,4-lutidine (pK, 7.0) when studies with both vanadyl cation and vanadate are to be carried out. Buffers such as triethanolamine (TEA), Bis-Tris propane (BTP), Tris, glycylglycine, Tricine, AMPSO, and
CRANS
b
ET
AL.
25
0.2 [v(Iv)],,,110-6
0.4
0.6 M
FIG. 2. (a) V,,/Vi ratios for the p-nitrophenyl phosphate hydrolysis catalyzed by alkaline phosphatase are plotted against total vanadate concentration (+), vanadate monomer concentration (m), vanadate dimer concentration (O), vanadate tetramer concentration (0), and vanadate pentamer concentration (A). The concentrations of various vanadate derivatives were determined using 51V NMR spectroscopy (+5% error limit) and the rates of hydrolysis were determined in 20 mM Hepes, 0.966 M KCl, 0.25 mM p-nitrophenyl phosphate at 23°C and pH 8.0 using from 0.007 to 0.04 mg/ml alkaline phosphatase. The correlation coefficient of the linear relationship between V,/Vi and monomeric vanadate was >0.995. (b) V,/V, ratios in various buffers are plotted against varying vanadate concentrations. The buffers are 50 mM Hepes ( X ), 50 mM Bis-Tris propane ( + ), 50 mM Hepes and 5 mM Tricine (A), 50 mM 2,4-lutidine (+), 50 mM N-ethylmorpholine (m), 50 mM barbitol, purged (0), 50 mM barbitol, unpurged (Cl). The correlation coefficients were all 0.99 or above. The enzyme rates were determined in the specified buffer, 1 M KCl, and 0.025 mMp-nitrophenylphosphate at 25°C and pH 8.0 using approximately 0.005 mg/ml alkaline phosphatase. (c) Vo/Vi ratios in various buffers are plotted against varying vanadyl cation concentrations. The buffers are as follows: 50 mM N-ethylmorpholine and 1 M KC1 ( X ), 50 mM N-ethylmorpholine and 0.5 M KC1 (0), 50 mM N-ethylmorpholine and 2.0 M KC1 (*), 50 mM barbitol (m), 50 mM Hepes (A), 50 mM Bis-Tris propane (A), and 50 mM 2,4-lutidine (0). The enzyme rates were determined in the specified buffers, in well-purged solutions, and with 1 M KC1 and 0.025 mMp-nitrophenyl phosphate at 25°C and pH 8.0 using approximately 0.005 mg/ml alkaline phosphatase.
DIPS0 should be avoided if both vanadium(IV) and (V) are to be measured. Most of these buffers form significant amounts of stable complexes with vanadate and/or vanadyl cation. Proportionality between V,lVi and Inhibitor Concentration The linear relationship between the V,/Vi ratio and the inhibitor concentration was examined experimentally for both vanadate and vanadyl cation (Figs. 2a-c). The inhibition by vanadate was first determined under conditions where the vanadate solutions consisted of complex mixtures of vanadate monomer, dimer, tetramer, and pentamer. The enzyme-catalyzed hydrolysis rates (Vi and VJ ofp-nitrophenyl phosphate (0.025 mM) were measured at 405 nm at vanadate concentrations ranging from 0 to 0.5 mM. The hydrolysis rates were measured with 0.007 to 0.04 mg/ml alkaline phosphatase in 20 mM Hepes and with 1 M KC1 (pH 8.0 at 25”C), and the V,/Vi ratios were calculated from these measurements. The concentrations of the various vanadate species in the assay solutions were also determined using 51VNMR spectroscopy. The details of these experiments are described under the Experimental Section. Plotting the V,/Vi ratio as a function of various vanadate derivatives shows a linear relationship with the vanadate monomer (R > 0.995) and nonlinear relationships with the vanadate dimer, tetramer, and pentamer. These re-
sults show the vanadate monomer is the only vanadate species in the solution that inhibits alkaline phosphatase significantly under the assay conditions. The inhibition by vanadate was also measured from 0 to 0.002 mM. Since the vanadate oligomerization reactions are in rapid equilibrium, the oligomers will always be present in solution; however, their concentrations will be very small. In the concentration range from 0 to 0.002 mM the vanadate monomer is for all practical purposes the only vanadate species present. The hydrolysis rates were measured in different buffers including Hepes, N-ethylmorpholine, 2,4-lutidine, barbitol, and BTP (Fig. 2b). The reaction rates were changed by purging the assay solution with nitrogen; however, these changes did not affect the V,/Vi ratio. Hepes has higher Vo/Vi ratios for vanadate than either N-ethylmorpoline, barbitol, BTP, or 2,4-lutidine, suggesting that these buffers interact more with vanadate than Hepes does. The inhibition by vanadyl cation was measured from 0 to 0.0007 mM vanadyl cation by monitoring the hydrolysis of p-nitrophenyl phosphate (0.025 mM). The reactions were carried out at several concentrations of barbito1 (20, 50, 67 mM) and in other buffers (50 mM Hepes, 50 mM 2,4-lutidine, 50 mM N-ethylmorpholine, and 50 mM BTP). Calculating the V,/V, ratios and plotting them as a function of the vanadyl concentration led to Fig. 2c. The curves in Fig. 2c show an initial linear region where the V,JVi ratio is proportional to the vanadyl cation concentration. As the V(W) concentration in-
KINETIC
. 0
METHOD
.
.
20
40
FOR
DETERMINATION
. 60
80
11 (Km + [S]) I lo3 w’ FIG. 3. The V,/lOV, ratio for vanadate (B) and the Vo/Vi ratio for vanadyl cation (Cl) are plotted as a function of l/(K,,, + [S]). The intercept on the Y-axis is l/lOK, or l/K,, respectively. The rate ratios for vanadate were determined in 20 mM Hepes, 0.966 M KCl, and 0.0025 to 0.50 mM p-nitrophenyl phosphate at 23°C at pH 8.0 using 0.0065 mg/ml alkaline phosphatase. The vanadate concentration was 0.010 mM. The correlation coefficient of the line was r0.995. The rate ratios for vanadyl cation were determined in 50 mM purged harhitol, 1 M KCl, and 0.0025 to 0.50 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase. The vanadyl cation concentration was 6.7 X 10-s M. The correlation coefficient of the line was 0.98.
creases, the concentration of free vanadyl cation as determined from the V,/Vi ratio levels off. Vanadyl cation is known to form oligomers or polymers at higher concentrations (25,26) and is therefore not expected to increase. Our observations are therefore consistent with previous reports suggesting that the maximum concentration of vanadyl cation is approximately 10m7M at neutral pH (25,26). Increasing the total vanadium concentration above 10m7 M will increase the overall vanadium(IV) concentration but will barely affect the free vanadyl cation concentration. Figure 2c also shows a noticeable decrease in the Vo/Vi ratio as the ionic strength increases. The proportionality factor q changes from 3.6 in 0.5 M KC1 to 2.5 in 1.0 M KC1 and to 1.5 at 2.0 M KCl. Consequently V,/Vi determinations at higher ionic strength will be more prone to error because of the smaller proportionality factor.
OF
FREE
V(W)
AND
59
-(V)
ear relationship as predicted in Eq. [2] is observed (R > 0.995). Corresponding experiments were conducted with 6.7 X lop8 M vanadyl cation using 50 InM barbitol as buffer. The V,/Vi ratios calculated from the observed rates are also plotted as a function of l/(K, + [S]) for vanadyl cation in Fig. 3. A linear relationship is also observed for this inhibitor (R = 0.98). We therefore conclude that the relationship shown in Eq. [2] is valid for both vanadate and vanadyl cation under the conditions examined in this work. Variations in the VO/V, Ratio as a Function of pH, Alkaline Phosphatase Concentration, and Temperature The changes in the V,/Vi ratios for vanadate, phosphate, and vanadyl cation were determined as a function of pH, enzyme concentration, and temperature. The hydrolysis rates ( Vi and V,) of p-nitrophenyl phosphate (0.025 mM) were measured from pH 7.8 to 8.5 with no inhibitor, with 0.010 mM vanadate, and with 0.30 mM phosphate. The hydrolysis rates were measured in 20 mM Hepes and 1 M KC1 (pH 8.0 at 25”C), and the V,/Vi ratios were calculated from these experiments. Corresponding experiments were carried out with vanadyl cation at 6.7 X 1O-8 in 50 mM barbitol. Plotting the V,/Vi ratios for vanadate, phosphate, and vanadyl cation as a function of pH shows that a considerable change in V,,/ Vi ratio takes place in the pH region under examination (Fig. 4). It is therefore important to maintain constant pH when determining V, and Vi, and we recommend keeping pH constant to within 0.05 pH unit. The variation of the Vo/Vi ratio as a function of enzyme concentration was determined with vanadate (0.010 mM), phosphate (0.30 mM), and vanadyl cation
+
-D V./IO”, (V(V))
“.I”1 ww *
(P,)
V./IO",
1.50 c. 2
1.25‘
co 1.00’
Dependence of the V,lVi Ratio on the Alkaline Phosphatase Substrate Concentration The linear relationship between the Vo/Vi ratio and the substrate (p-nitrophenyl phosphate) concentration was examined experimentally for vanadate and vanadyl cation. The hydrolysis rates (Vi and V,,) ofp-nitrophenyl phosphate (0.025 mM) were measured at substrate concentrations from 0.0050 to 0.50 mM in the presence of no inhibitor and in the presence of 0.010 mM vanadate. The hydrolysis rates were measured with 0.006 mg/ml alkaline phosphatase in 20 mM Hepes and with 1 M KC1 (pH 8.0 at 25”C), and the V,/Vi ratios were calculated from these measurements. The Vo/Vi ratios were plotted as a function of l/(K, + [S]) for vanadate in Fig. 3. The lin-
3
0.75
s
0.50
2
0.25’
i7
-
0,ooi
7.6
I 7.8
-
I 8.0
-
I 8.2
-
I 8.4
8.6
PH FIG. 4. The V,,/V, ratio for vanadyl cation (+) and the V,,/lOV, ratios for vanadate (0) and phosphate (w) are shown versus pH. The V,/lOV, ratios for 0.010 mM vanadate and 0.30 mM phosphate were determined in 20 mM Hepes, 0.966 M KCl, and 0.025 mMp-nitrophenyl phosphate at 23°C and pH 8.0 using 0.007 mg/ml alkaline phosphatase. The V,,/V, ratio for 6.7 X lo-* M vanadyl cation was determined in 50 mM purged barbitol, 1 M KCl, and 0.25 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase.
60
CRANS TABLE
V,/Vi Vanadyl tions
Ratios Cation
Determined at Various
1
for Vanadate, Phosphate, and Alkaline Phosphatase Concentra-
AL.
preincubation of the assay solution) when measuring VO, Vi 9Vo,ref7and Vi,refV,lVi Ratio Determination in a Solution Containing Multiple Components
VOlvi
[API
ET
X lo3 (mg/ml) 1.32 1.8 2.2 2.6 2.7 3.1 3.5 3.8 4.2 4.4 5.2 6.2
Average Standard deviation 95% confidence limit a The V, was determined in mM p-nitrophenyl phosphate mined with 0.010 mM vanadate. b The V, was determined in mM p-nitrophenyl phosphate mined with 0.30 mM phosphate. ’ The V, was determined in mM p-nitrophenyl phosphate mined with 6.7 X 10-s M vanadyl
VW)
a
Pb
11.4 -
2.86
11.7 10.8
3.00 2.95
11.6 11.8
2.94 2.99
11.4 0.40 0.19
2.95 0.055 0.026
V(IV)C 1.16 1.16 1.13 1.20 1.18 1.14 1.11
1.15 0.030 0.040
20 mM Hepes, 0.966 M KCl, and 0.025 at pH 8.0 and 23°C. The Vi was deter20 mM Hepes, 0.966 M KCI, and 0.025 at pH 8.0 and 23°C. The Vi was deter50 mM barbitol, 1.0 M KCl, and 0.025 at pH 8.0 and 25°C. The Vi was detercation.
(6.7 X lo-’ M) by measuring the hydrolysis rate of p-nitrophenyl phosphate (0.025 mM). The vanadate and phosphate measurements were conducted in 20 mM Hepes and the vanadyl cation experiments in 50 mM barbitol. The enzyme concentration varied from 0.002 to 0.007 mg/ml, and the resulting rates were used to calculate the Vo/Vi ratios. The results are given in Table 1. Changing the enzyme concentration caused no apparent change in the V,/Vi ratio. The concentration of alkaline phosphatase used in the assays should therefore not affect the Vo/Vi ratio as long as both V, and Vi are determined accurately. The variation of the V,/Vi ratio as a function of temperature was determined for vanadate at 0.002 and 0.010 mM (Table 2). The V,,/ Vi ratio changed from 2.85 at 20°C to 2.42 at 35°C for 0.002 mM vanadate. At 0.010 mM vanadate the Vo/Vi ratio changed from 9.30 at 20°C to 7.60 at 35°C (Table 2). Temperature changes are therefore likely to affect the Vo/Vi ratio significantly. Even a 1-2” change caused by adding a cool substrate solution into an incubated assay solution could affect the V,/Vi ratio by 5510%. We therefore recommend that care be taken in carrying out the enzyme assay using identical experimental technique (preferably including 2 min or longer
The linear relationship between V,lVi and vanadate monomer concentration should not be affected by additional substrates and inhibitors in the assay mixture (as predicted by Eqs. [6]-[8]). Such relationships were examined by measuring the rates of hydrolysis at varying concentrations of vanadate in the presence of 0.025 mM p-nitrophenyl phosphate and an additional inhibitor (0.5 mM phosphate) or enzyme substrate (0.5 mM glycerol 3-phosphate). The V,/ Vi ratios were calculated from the rates and plotted as a function of vanadate concentration and are shown in Fig. 5. Linear relationships between the V,/Vi ratio and the vanadate concentration are observed in all cases although the presence of additional inhibitors and/or substrates in the assay solutions decreased the proportionality factor, q. Application of the Alkaline Phosphatase Assay In biological systems vanadium readily converts from oxidation state V to IV and vice versa. Since the oxidation state of vanadium is crucial to how vanadium acts, it is of interest to determine the concentrations of both free oxidation states IV and V. In this section we demonstrate how either V(N) and V(V) can be determined in solutions containing either or both V(IV) and V(V). The Vo/Vi ratio expresses the concentration of both free V(V) and V(IV), if the V, is determined in the presence of the sample and a complexing agent of both vanadium(N) and (V) (such as DIPS0 or Tricine) (20). All components in the sample will affect the V,, in a manner similar to Vi, and thus only the concentration of V(V)
TABLE
Vo/Vi Ratios Vanadate
Determined at Various
2 for 2 X lo-’ and Temperatures”
10e5 M
VolK Temperature (“0
20 22.5 25 27.5 30 32.5 35
2 x 10-6M
1o-5
VW
VW)
2.85 2.79 2.64 2.48 2.46 2.48 2.42
a The V, was determined in 50 mM Hepes, 1 M KCl, p-nitrophenyl phosphate at pH 8.0 using approximately alkaline phosphatase.
M
9.30 8.78 8.77 8.19 8.14 7.88 7.60 and 0.025 mM 0.005 mg/ml
KINETIC
5 ‘0 >
METHOD
FOR
DETERMINATION
15.
1.0
0.5 0.0
1.0
[V(V)]
2.0
11O-6 M
FIG. 5. The V,/V, ratios in the presence of various additional inhihitors, substrates and complexing agents are shown for vanadate. The additional compounds are: none ( + ), 2.5 mM BTP (*), 10m7 M vanadyl cation (+), 0.5 mM glycerol 3-phosphate (W), 0.5 mM phosphate (O), 0.5 mM glycerol 3-phosphate and 0.5 mM phosphate (A), 2.5 mM BTP and 10m7 M vanadyl cation ( X ), and 5 mM Tricine (A). The V,/V, ratios were determined using the above components, 50 mM Hepes, 1 M KCI, and 0.025 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase. The correlation coefficients were 0.99 or above unless specified otherwise.
and V(IV) will dictate the V,/Vi ratio according to Eq. [8]-[lo]. Similarly, the V,/Vi ratio could be used to measure the concentration of free V(W), if V, is determined in the presence of the sample and a V(W)-specific complexing agent such as BTP. The Vo/Vi ratio could also be used to measure the concentration of V(V) if V,, is determined in the presence of a complexing agent specific for V(V). This approach was demonstrated by measuring the V,/ Vi ratios for a solution containing a mixture of vanadate and vanadyl cation. The rates of hydrolysis ( V, and Vi) of p-nitrophenyl phosphate were measured at varying vanadate concentrations in the presence of 1.3 X 10e7 M vanadyl cation (approximately 35% inhibition) and a linear relationship was observed when plotting the V,/ Vi as a function of [V(V)] (Fig. 5). The rates obtained in the presence of Tricine, vanadyl cation, and various concentrations of vanadate should therefore be identical with a V,, obtained in the absence of either vanadate or vanadyl cation, and this was indeed observed (Fig. 5). A corresponding series of rate measurements carried out in the presence of 1.3 X 10e7M vanadyl cation, BTP, and varying vanadate concentrations again yielded a linear relationship with a nonzero proportionality factor q. This is in accord with the expectation that BTP selectively complexes vanadyl cation. By combining the measurements using both BTP and Tricine, the V(IV) concentration, the total vanadium concentration, and the V(V) concentration can be calculated. The Vo/Vi ratio was determined in barbitol in the presence of 0.001 mM vanadate with varying concentra-
OF
FREE
V(IV)
AND
-(V)
61
tions of vanadyl cation. A linear relationship was observed between the V,/Vi ratio and the [VO”‘] at low concentrations and is shown in Fig. 6. The rates obtained in the presence of BTP and varying vanadyl concentrations should be identical with the rate obtained without vanadyl cation but with 0.001 mM vanadate. This was indeed observed. The Vo/Vi ratios determined in the presence of 5 mM Tricine were 1.0 in accord with the expectation that Tricine would complex this range of vanadyl cation concentrations. An attempt to determine the free vanadyl cation concentration in the presence of 0.5 mM phosphate was complicated by the fact that phosphate complexed the vanadyl cation. As a result very little (less than 0.03 X lo-’ M) of the original (from 0.03 to 0.7 X lop8 M) vanadyl cation was free in these assay solutions containing phosphate. Glycerol 3-phosphate (0.5 mM) was also found to complex the vanadyl cation but much less than phosphate. The proportionality factor q in a plot of V,/Vi as a function of vanadyl cation concentration was >l (fO.l) in the presence of 0.5 mM glycerol 3-phosphate, suggesting that not all the vanadyl cation was complexed by glycerol 3-phosphate. The variation in vanadyl cation concentration in the presence of glycerol 3-phosphate can be determined using the assay. These experiments illustrate the ability of many biomolecules to complex vanadyl cation. Complex fluids with high concentrations of vanadyl-complexing compounds are therefore likely to contain both the complexed vanadyl cation and only little free vanadyl cation (25,26). In order to determine the concentration of free vanadyl cation in such a biological sample containing complexing agents, such agents must be present when measuring the proportionality factor.
0.84
0.0
-
m
0.2 [V(lV)]tot
0.4
0.6
/l O-6 M
FIG. 6. The V,,/Vi ratio in the presence of various additional inhibitors, substrates and complexing agents are shown for vanadyl cation. The additional compounds are: none (A), 2.5 mM BTP (O), 10m6 M vanadate ( + ), 0.5 mM glycerol 3-phosphate (m), 0.5 mM phosphate (+), 10m5 M vanadate and 2.5 mM BTP (O), and 10m6 M vanadate and 5 mM Tricine ( X ). The Vo/Vi ratios were determined using the above components, 50 mM barbitol, 1 M KCl, and 0.025 mM p-nitrophenyl phosphate at 25°C and pH 8.0 using 0.005 mg/ml alkaline phosphatase.
62
CRANS
The described method is applicable when Km, V,,,,,, are constant under the various rate determinations. Such assumptions are nontrivial with alkaline phosphatase, because alkaline phosphatase has Michaelis-Menten constants that are extremely sensitive to the environment. Variations in buffer, ionic strength, temperature, and pH have been shown to change the Km and Ki for vanadate by several orders of magnitude (17,X3). Using the V,/Vi ratio to determine [IleR, one can circumvent the problem of the extreme sensitivity to environment by using the standard addition technique (32). The experimental determination of V, and VO,ref will require a complexing agent that will specifically bind the species of interest. We have used BTP to selectively complex vanadium(N) because this buffer will complex vanadium(rV) in the concentration range 10-s to 10e6 M without binding vanadium(V) in the concentration range 10e7 to 10e5 M. Other sequestering agents could substitute for BTP if this buffer causes problems in any particular samples. If a substitute for BTP is needed, a potential candidate should not change the V, of a reference buffer more than 1620%. This requirement is based on the observations that the rates of the reaction catalyzed by alkaline phosphatase are related to the selectivity with which alkaline phosphatase binds substrate and/or inhibitor. If no changes in the reaction rates are observed in the presence of the ligand, the ligand does not appear to affect the Vo/Vi ratio. Quantitative determination of vanadium concentrations can therefore be obtained by measuring Vo, Vi, VO,ref, and Vi,refp where V, and VO,re.are determined in the presence of the specific sequestering agent, Vi and Vi,ref are determined without such a compound, and a known concentration of vanadium is added
Ki, and KJK,
to
Vi,ref*
The sensitivity of alkaline phosphatase to its environment may render this assay inappropriate for quantitative determinations with some samples. The following control experiment will demonstrate whether such conditions indeed prevail. The experiment involves the determination of the V,/Vi ratio for an innocuous (to the sample) competitive inhibitor that is known not to be present in the sample under investigation. The corresponding V,/Vi ratio of this inhibitor in the presence of the biological environmental sample is also determined. Examples of such inhibitors are phosphate, molybdate, arsenate, and tungstate. If the two V,/Vi ratios are identical, the assay conditions are compatible with the biological sample. On the other hand, if the V,/Vi in the presence of biological or environmental sample is significantly larger or smaller (> or
