ANALYTICAL BIOCHEMISTRY 68, 1-8 (1975)
Polarographic Determination of Superoxide Dismutase ADELIO RIGO, PAOLO VIGLINO
Institute of Physical Chemistry, University of Venice, Venice, Italy AND GIUSEPPE ROTILIO
Institute of Biological Chemistry, University of Rome, Rome, Italy Received December 23, 1974; accepted March 3, 1975 A polarographic procedure is described which allows determination of the catalytic constants for superoxide dismutase-catalyzed reactions. The method presents a single and rapid evaluation of the enzyme concentrations as well as determination of its activity under different conditions; e.g., p H between 9 and 13, presence o f urea, guanidine, sodium dodecyl sulphate and inhibitors such as C N - and N3-. The results fit very well with data previously obtained with other methods and show that this polarographic procedure can be used under conditions that render the other methods unsuitable for the measurement of the enzyme activity.
The superoxide radical ion 02% intermediate in the reduction of molecular oxygen, is directly or indirectly involved in a series of biochemical reactions and biological phenomena such as lipid peroxidation, enzymatic hydroxylations, bacterial killing etc. Superoxide dismutases are a class of metalloenzymes that are present in all aerobic organisms and control the concentration of 05- in the cell since they accelerate the rate of the reaction 202- + 2 H +
) H 2 0 2 -~- 0 2
by many orders of magnitude. All the analytical methods for the determination of superoxide dismutase are based on this ability to accelerate the dismutation of 02- and require a source of superoxide ion and a system for detecting it. Both these requirements are routinely satisfied by methods that involve additional components and are quite indirect. A review of these methods is reported by Fridovich (1). In particular, xanthine oxidase oxidizing xanthine (2, 3), N A D H in the presence of phenazine methosulphate (4), photoreduced dyes (5) have been used as source of 02- in aerated aqueous solutions. Furthermore 02- generated electrolytically by the 1 Copyright© 1975 by AcademicPress, Inc. All rightsof reproductionin any formreserved.
RIGOr VIGLINO AND ROTILIO
cathodic reduction of 02 has been shown (6) to diffuse into aqueous solutions to an extent which allows reactions with small molecules such as epinephrine and with superoxide dismutase. For the detection of unreacted Oz-, the reduction of either cytochrome c (2, 3) or nitroblue tetrazolium (4, 5) or the oxidation of epinephrine (2) by 02- has been employed. By these methods the superoxide dismutase concentration or activity is determined indirectly from its capability to inhibit the reaction of 02- with the detector system. Furthermore some assays are based upon autoxidation. In such assays the autoxidizing substance, e.g., epinephrine (7), pyrogaUol (8), serves both as the source of O2- and as the indicating scavenger for 02-. Since these methods are based on a series of consecutive and parallel reactions, they do not permit a direct determination of the kinetic parameters; furthermore they do not give a linear response to increasing enzyme concentrations and produce O2- at a very low steady-state concentration. A direct method is achieved when pulses of energetic electrons are directed into oxygenated aqueous solutions, in the presence of suitable scavengers, to generate spectrophotometrically detectable levels of 02- (9, 10). This technique meets the demands of the ideal method for both determination of enzyme activity and investigation of the reaction mechanism; that is, 1) direct determination of the rate constant; 2) high sensitivity; 3) linear dependence on enzyme concentration; 4) high yield of O2-- Points 1, 2, and 3 allow safe determination of enzyme activity, while point 4 permits mechanism studies. On the other hand, pulse radiolysis requires very expensive and special apparatus. A method which appears to satisfy the demands of the ideal one and to require routinely available instrumentation as well is the kinetic currents technique. In this method, the dropping mercury electrode (DME) acts in an O2-satured aqueous solution as a source of 02and a detector of the rate of its dismutation as well. The method will be described in the following report and some of its applications to current problems of superoxide dismutase biochemistry will be presented. EXPERIMENTAL PROCEDURE t. Materials and instrumentation. Bovine superoxide dismutase was prepared from bovine red blood cells according to McCord and Fridovich (2). All solutions were prepared from analytical grade chemicals dissolved in twice-distilled water and air saturated at the indicated temperature. Measurements were made in a three-electrode polarographic cell fitted with a dropping mercury electrode, a platinum counter electrode and a satured calomel electrode (SCE) as reference. An A M E L polarographic unit, Model 461, and an A M E L pH meter,
POLAROGRAPHIC DETERMINATION OF SUPEROXIDE DISMUTASE
Model 331 were employed for the polarographic and t he pH measurements, respectively. 2. Procedure for measurements. An enzyme concentration ranging between 10-10 and 10-9 M- was realized by subsequent additions of superoxide dismutase to a buffered solution, usually sodium borate, 0.025 M, and at least 2 × 10~4 M in triphenylphosphine oxide (TPO): After each addition, t h e height of the polarographic wave was recorded at a fixed potential, usually - 1 V vs SCIE. The wave height corresponding to the complete dismutation of O~7- in the reaction layer was obtained either by adding a relatively concentrated superoxide dismutase solution (10-7 M in the cell) or by adding few drops of a 5 M H2SO4 solution to obtain a pH in the range 2,-5. 3. Theory. The electroreduction of molecular oxygen in aqueous solutions at the D M E is a bielectronic process since the 0~= genera:tedin a,, first step is immediately reduced, in the presence of a source of protons; according to the generally accepted: mechanism (11): O~+e. ')02-, 02- + H30 + ~ HOE + H20; HOt + e ,~HO~=, H O U + H '+ , H20~. I t has recently been demonstrated- that in the presence of a hydrophobic surfactant,, such a s o~-quinoline (t2) or triphenylphosphinoxide (TPO) (13), able t o cover t he electrode surface with a monomolecular I~A
~" i-t: hi+h2
-1.5 V E/SCE
FIG. 1. Polarographic waves of Oh in sodium borate 0.025 m; temperature 23°C. Curve a, pH 12.5; TPO, 9 x 10-4 M; b, pH 9:9; TPO, 9 × 10-4 M; c, pH 9.9; TPOi 9 × 10-4 M, superoxide dismutase, 1.5 × 10-9 M; d, p H 9.9; the same curve is obtained at pH 9.9 in the presence of TPO and superoxide dismutase at 10-r M.
RIGO, VIGLINO AND ROTILIO
layer, the proton transfer to O2- on the surface of D M E is inhibited, and as a consequence the reduction of 02- is prevented. In Fig. 1 are reported the plots of the cell currents vs the potential for the reduction of 02 either in the presence or in the absence of a surfactant, to which correspond the processes 02 + e ) O2- (curve a) and 02 -4- 2e -4- 2 H ÷ ~ H202 (curve d). The value of the limiting current at --1 V is controlled by the diffusion of the O2 to the electrode surface, and as a consequence it is proportional to the concentration of 02 in the solution. The addition of a compound able to catalyze the dismutation of O2- into H202 and 02, i.e., H +, superoxide dismutase etc. converts part of the 02- present in the reaction layer to O2 which, adding to the diffusive stream of O2 from the solution to the D M E , will increase the limiting current. Under these conditions the D M E is at same time a source of 02- and a very sensible detector of its dismutation rate. The reaction scheme for such a process is O2+ e 2 0 2 - q- C
~ O2-, ) 02 q-- H202 q- C
where C is a catalyst. According to K o u t e c k y and co-workers (14), the exact solution of the kinetic differential equations for a similar process may be replaced, with an error of less than I%, by this simplified function: t,/Ta = (1/7.42 + 1.25 X1),
if X1 ranges between 0 and I0, where X1 = k[C]tg, T1and To are the mean limiting currents either in the presence or in the absence of C respectively, tg the drop time of mercury electrode and k the rate constant of the reaction . Since 02- partially dismutates in the reaction layer to an extent which is p H dependent (15), T~and To are measured, as reported in Fig. 1, to account for this side reaction. Equation  can be rewritten in the form ((7.42 T~/Ta)-- 7.42)/(2.25 -- (~/T,1)) = k[C]tg,
where the left side is a linear function of superoxide dismutase concentration for X1 values below 10.
RESULTS A N D DISCUSSION
Calculation of k and Enzyme Concentration Figure 2 shows a plot of the experimental data as a function of the superoxide dismutase concentration according to Eq. . A very good fit is obtained. F r o m the slope of this plot a k value of
POLAROGRAPHIC DETERMINATION OF SUPEROXIDE DISMUTASE
F~o. 2. T h e straight line r e p r e s e n t s a plot of the left side of Eq.  vs e n z y m e concentration; sodium borate, 0.025 M; T P O , 9 × 10 4 M; p H = 9.8; t g = 2.9 sec; t e m p e r ature 25°C.
2.30__+ 0.07 x 109 M-1 sec -~ was obtained at 25°C and / x = 0.1, which agrees quite well with the value calculated from pulse radiolysis, 2.30 x 109 M-1 sec -1 (9, 10), and from kinetic competition methods, 2.1 × 109 M-a sec -a (16). Conversely, assuming the value of the kinetic constant k, the superoxide dismutase concentration can be easily calculated. With a k value of 2.3 x 109 M-~ sec -~ and tg of 3 seconds, superoxide dismutase concentrations between 10 -1° and 1.5 x 10 -8 M give linear increases of the left side of Eq. . As a consequence, in this range of concentrations the determination of superoxide dismutase is direct and does not require calibration measurements. The reproducibility of these determinations is quite good; in fact, at a constant p H a mean standard deviation of 3% was calculated.
Determination of Enzyme Activity under Different Conditions We examined by this polarographic method the effect on the e n z y m e of a number of factors that had already been tested by other methods with respect to their influence on superoxide dismutase activity. Potential denaturants such as urea, guanidine and sodium dodecyl sulphate (SDS) have been used in the epinephrine oxidation assay (17) to test the stability of the bovine enzyme. Because of the importance of these agents in studies of enzymes, we investigated the suitability of the polarographic method in their presence. SDS, guanidinium chloride and urea do not modify the polarographic wave in the potential region where 02- is produced. F u r t h e r m o r e from the activity measurements in the
RIGO, VIGLINO AND ROTILIO
presence,Of these c o m p o u n d s it results that :SOS concentrations as high a s 0.15,M d o n o t c h a n g e the activity of the bovine superoxide dismutase while, in the highly -viscous 3 and 10 M u r e a solutions, the activity o f l h e ,enzy~'ne is qo~ered b y a b o u t 35 and 45%, respectively, which c o n ~ : r ~ t h e d a t a reported by ,Forman and Fridovich (17). Finally, t h e :decrease o f activity o f :the bovine superoxide dismutase o b s e r v e d in the ~presence ,of guanidium chloride c a n :be attributed 'to t h e effect of C 1 - a n d ~ofionic ,strength ,(18) which means there is :not ,a specific inhibition by :this compound. M o r e o v e r C N - and N~- :~ere s h o w n t o interact with the copper of bovine superoxide dismutase by electron paramagnetic r e s o n a n c e (19). Pulse ,radiolysis m e a s u r e m e m s demonstrated that C N - inhibited the enz y m e powerfully, while n o effect was seen with N3-, t0 -3 M. B y the polarographic m e t h o d t h e ;kinetic constant of O~- dismutation as a function of the concentrati0n,of C N - and Na- was measured. The results obtained ~together with the relative inhibition constants K~ are reported in Table 1. The data show t h a t in both :cases ,the inNbition is r e l a t e d t o the interaction of the anion wi~h ~the a c t i , e ,metal. M o r e o v e r they s h o w that the polarographic method is suitable in c a s e s where pulse :radiolysis c a n n o t b e used, as in the N a- inhibition experiment :in which the concentration of inhibitor cannot :he raised above |0=aM because of the formation of the intense optical absorption :band of the c o p p e r - a z i d e complex. Since the mean limiting current relative to the reduction of O2 to O2changes :wRh the pH, the activity of superoxide dismutase was measured as function of H + concentration in the p H range 9 - 1 3 . The results, TABLE
~NHIBITION OF SUPEROXIDE DISMUTASEa
Inhibitor Ty~pe CNCNCNCNN~N3N3N3
Concentration (M) 0,62 x 1,99 x 6,76 x 27:5 x 0.75 × 1.25 x 2.5 x 5.0 x
10-6 10-6 10-~ 10-6 10-2 10-2 10=2 t0 -2
kb (M-1 sec-1 × lO~9)
1,69 1.08 0.52 0.13 1:98 1.57 0.77 0.'59
1.72 × 10-6 1.76 × 10-6 1:97 x 10-6 1,64 × 10-6 1.67 × 10-z 1.52 x 10-z 1.25 x 10-2 1.29 × 10-~
1 . 7 7 × 10 -6
1 . 4 3 x 10 . 2
a The ,measurements were done in sodium borate buffer, 0.025 M at pH 9.80 and temperature,of 25~C. b Kinetic constants of 02 dismutations :by bovine superoxide dismutase in presence of inhibitors,
P O L A R O G R A P H 1 C D E T E R M I N A T I O N OF S U P E R O X I D E D I S M U T A S E
F16. 3. pH dependence of the enzyme activity; sodium borate, 0.025 M (O); sodium phosphate, 0.05 M (A); temperature, 25°C. reported in Fig. 3, show a decrease of the activity above p H 10. Between p H 10 and 12.5 this decrease was shown to be reversible by lowering the p H below 10, while above p H 12.5 there was an irreversible loss of activity due to the alkali denaturation of the protein. T h e reversible decrease of activity between p H 10 and 11,3 was already observed with pulse radiolysis measurements (20). As regards the influence of temperature, no dependence of superoxide dismutase activity on this parameter was evident in the range 0-37°C. This result does not agree with the fact that by pulse radiolysis a small but still measurable activation energy was found (21). It cat~ be concluded that the kinetic current method offers the possibility of a rapid, accurate and reproducible determination o f superoxide dismutase concentration and activity. It also appears v e r y suitable to the study of this e n z y m e at the molecular level because it allows the testing of the effects various factors have, effects which would interfere with the other components of the systems routinely used to assay the activity and even with the pulse radiolysis measurements. Finally owing to the absence of interfering reactions and the linearity of the response, the method may be very useful for the determination of superoxide dismutase concentrations in biological fluids. Further potentialities o f the method will be the object o f a separate report.
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RIGO, VIGLINO AND ROTILIO
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