Photnchemisrrj mid Photnhiologv, 1Y7h. Vol. 23, pp. 155-161.
Pergamon Press. Printed in Great Britain
THE MECHANISM O F ENERGY TRANSFER FROM POLY-I)-BENZOYLPHENYLACETIMIDO-BOVINE SERUM ALBUMIN TO SMALL-MOLECULE QUENCHERS PATRICK S. MARIANO,*GEORGE I. GLOVER*and TIMOTHY J. WILKINSON Department of Chemistry, Texas A & M University, College Station, TX 77843, USA. (Received 17 July 1975; accepted 16 October 1975)
Abstract-Results of a quantitative photochemical study of poly-p-benzoylphenylacetimido-bovine serum albumin in the presence of small-molecule triplet quenchers are reported. The efficiency of quenching by organic salts containing low triplet energy chromophores is shown to be qualitatively dependent on their predicted association constants to the modified protein. In addition, quenching is inhibited by salts of organic acids which possess high binding affinities for the protein but do not contain chromophores of low triplet energy. Quantitative treatment of the quenching and inhibition data yields results which strongly support the operation of an ‘affinity controlled’ mechanism for triplet energy transfer from the benzophenone moieties of the modified-bovine serum albumin to quenchers such as cc-naphthylacetate and trans-cinnamate.
INTRODUCTION
In the preceding paper (Mariano et al., 1975) we reported an interesting photochemical reaction of poly-p-benzoylphenylacetimido-bovineserum albumin (mod-BSA), a modified protein containing benzophenone moieties covalently linked to the protein backbone through the &-aminogroups of lysine residues. Evidence was presented which indicated that the excited state reactions occurring from the triplet excited benzophenone groups involve the photoreductive addition of the excited carbonyls to carbon-hydrogen bonds within the protein backbone resulting in the introduction of benzhydrol groups as crosslinks in the protein (see Fig. 1 for a schematic representation of the process). Evidence for the nature of the photochemical reaction came from the observation that sodium trans-cinnamate, containing the low triplet energy styryl chromophore (ET = 62 kcal/mol), effectively quenches the mod-BSA photoreaction as a result of its role as an acceptor in the triplet energy transfer process. Support for this view derived from the observation that mod-BSA serves as a photosensitizer of the wellknown (Bregman et al., 1964) cis-trans isomerization of the triplet cinnamic acid salt. Additional evidence to substantiate this conclusion was found using emission spectroscopy. Phosphorescence of the benzophenone chromophores in mod-BSA was efficiently quenched by sodium a-naphthylacetate with concomittant sensitization of naphthalene-like phosphorescence. *The authors to whom correspondence should be communicated. ?The terminology, ‘affinity controlled’, is suggested by the fact that energy transfer by this mechanism will have its efficiency controlled by the affinity of the protein sensitizer for the quencher.
Our studies of the mod-BSA photoreaction have led to further investigations designed to determine if an affinity controlledt (static) (Vaughan and Weber, 1970; Lehrer, 1971a, 1971b) or diffusion controlled triplet energy transfer mechanism is operating in those cases where quenching of the mod-BSA photoreaction was detected. In the former mechanism, energy transfer from the triplet excited benzophenone moieties occurs to quencher molecules while they are reversibly bound to the modified protein. We would like to report the results of this investigation, which strongly implicate the operation of an affinity controlled mechanism for triplet energy transfer from mod-BSA to quenchers such as the sodium salts of a-naphthylacetic and trans-cinnamic acids.
Figure 1. Schematic representation of the mod-BSA photoreaction which involves photoreductive addition of the benzophenone moieties to a-CH bonds within the protein backbone. MATERIALS AND METHODS
General. Poly-p-benzoylphenylacetimido-bovineserum albumin was prepared according to procedure reported earlier (Mariano et al., 1975). trans-Cinnamic acid (Sigma) was twice recrystallized prior to use. /3-Naphthyltrimethylammonium chloride was prepared from the corresponding iodide by anion exchange chromatography. All other compounds were commercially available and were of reagent grade or better. All,buffered solutions were made with the same pH 8.0 buffer consisting of 0.05M KH2P04 and 0.1 M KCI, adjusted to pH 8.0 with 5 N KOH. UV spectral analyses were accomplished using a Beckman Acta 111 UVvisible spectrophotometer.All simultaneous irradiations of
155
PATRICK S. MARIANO,GEORGE I. GLOVER and TIMOTHY J. WILKINSON
156
65-
Quencher concentration.
[QI,
mM
Figure 2. Plots of mod-BSA photoreaction quenching efficiencies (Vo/V,) vs quencher (Q) concentration for sodium r-naphthylacetate (@), trans-cinnamate (W), sorbate (W), octanoate (0)and 3-phenylpropionate (U), and trimethylP-naphthylammonium chloride (V)as quenchers. multiple samples were carried out using an apparatus consisting of a merry-go-round at the center of which was located a 450W Hanovia medium pressure lamp surrounded by a uranium glass filter (T330nm = 5%, &sonrn = 45%, T370n,,, = 76%) in a water-cooled quartz immersion well. All photolysis solutions were degassed in uacuo and saturated with N, prior to use. However, we have found that dissolved oxygen has no effect upon the rate of the modified-protein photoreaction. Irradiations of mod-BSA in the presence of’ varying concentrations of potential quenchers. Stock solutions of the following potential quenchers were prepared in pH 8 buffer: sodium a-naphthylacetate (59 mM), j-naphthyltrimethylammonium chloride (59 mM), sodium trans-cinnamate (59 mM), sodium sorbate (59 mM), sodium octanoate (59 mM) and sodium 3-phenylpropionate (59 mM). Aliquots of these solutions were diluted with appropriate amounts of buffer to give a series of solutions of varying concentrations. One md portions of these solutions were mixed with 2 md of a mod-BSA solution (65 p M in pH 8 buffer) to give final solutions containing the varying concentrations of the four quenchers as indicated in Fig. 2. These mod-BSA solutions along with a control consisting of mod-BSA alone were simultaneously irradiated for 2 h in the merry-go-round apparatus using uranium glass-filtered light. One m t of each photolysate was gel filtered on Sephadex G-25 (fine) to separate quenchers from protein. The protein-containing peaks were collected and diluted to 10.0me with pH 8 buffer. The absorbances at 260 nm of these solutions were used to obtain the relative observed mod-BSA reaction velocities according to the following method. During an independent and extensive irradiation of mod-BSA (7.2 pM), the absorbance at 260 nm decreased from its initial value of 0.988 (A,) and after ca.
6 h leveled at a value of 0.490 ( A z ) . A plot of the natural log of the mole fraction of benzophenone chromophores remaining at time 2, expressed as AO-A/Ao-A,, where A is the absorbance at t, vs time is linear throughout the duration of the irradiation. As a result, the absorbances of proteincontaining solutions, from the 2 h simultaneous irradiations in the presence of quenchers and after chromatographic separation, are inversely proportional to the observed mod-BSA reaction velocities. The results of these experiments are recorded in Fig. 2. Irradiation of mod-BSA in the presence of quenchers and inhibitors. Sodium a-naphthylacetatr and octanoate. Thirtytwo 3 mY solutions each containing mod-BSA (42.6 p M ) in sets of eight containing sodium a-naphthyacetate at concentrations of 0.0, 14.9, 37.0, and 74.1 mM were prepared. Each set of eight solutions of constant a-naphthylacetate concentration contained sodium octanoate in the following concentrations: 0.0, 2.46, 4.91, 7.36, 11.1, 14.8, 19.6 and 24.5 mM. The solvent in each case was pH 8 buffer. These were simultaneously irradiated in a merry-go-round apparatus with uranium glass-filtered light for 3 h. The photolysates were individually gel-filtered on Sephadex G-25 (fine); the protein-containing peaks were collected and diluted to a fixed volume and analyzed by ultraviolet spectroscopy to determine the observed mod-BSA photoreaction velocities. The results from this experiment in terms of relative observed reaction velocities for the mod-BSA are plotted in Fig. 3. Sodium trans-cinnamate and 3-phenylpropionate. Thirtytwo 3 mf solutions containing mod-BSA (42.6 p M ) , sodium trans-cinnamate (0.0, 13.2, 32.9, and 65.9 mM) and sodium 3-phenylpropionate (0.0, 24.6,4.91, 7.36, 11.1, 14.8, 19.6 and 24,5mM) prepared in the same way as described above in pH 8 buffer were simultaneously irradiated in a merrygo-round apparatus using uranium glass-filtered light for 3 h. Analyses of the extent and relative observed rate of the mod-BSA reaction were conducted in the same mannei as described above. Results of this experiment are recorded in Fig. 4. Sodium a-naphthylacetate and propionate. Thirty-two 3 m/ samples containing mod-BSA and sodium a-naphthylacetate, in the same concentrations as for the a-naphthylacetate/octanoate experiment and sodium propionate (0.0, 2.46. 4.91, 7.36, 11.1, 14.8, 19.6 and 24.5mM) were simultaneously irradiated and analyzed as described above. Results from this experiment are recorded in Fig. 5. RESULTS
Quenching of’ the mod-BSA photoreaction using a variety oforganic salts. In an attempt to first qualitatively evaluate the mechanism of triplet energy transfer from mod-BSA, excited at wavelengths greater than 320nm, an exploratory investigation of the effect of quenchers on the mod-BSA photoreaction was conducted. In Fig. 2 and Table 1 are summarized the
Table 1. Mod-BSA photoreaction quenching efficiencies for various low triplet energy quenchers
*
Quenchers
Triplet Energies (kcal/mol)
Relative Quenching Efficiency ?.
Sodium a-Napthylacetate
61.0
8.57
Sodium =-Cinnamate
61.5
3.77
Sodium Sorbate
59.5
1.15
Trimthy 1-B-naphthylammonium chloride
61.0
1.00
*Assumed on basis of chromophores present in salts. ?Based on the slopes of the Vo/V, vs [Q] plots, and setting that for the ammonium salt as 1.
Energy transfer from modified BSA to triplet quenchers results of these experiments in which the observed velocity of the mod-BSA photochemical reaction was measured as a function of concentrations of various organic salts. The sodium salts of a-naphthylacetic, trans-cinnamic, sorbic, octanoic, and 3-phenyl-propionic acids, and b-naphthyItrimethylammonium chloride were chosen on the basis of either their potential binding affinities to mod-BSA and/or their possession of low triplet-energy chromophores. Plotted in Fig. 2 are the ratios of the observed photochemical reaction velocities in the presence and absence of these compounds (Vo/V,)vs their concentrations,
CQJ Quenching of the mod-BSA photoreaction in the presence of competitive inhibitors. Definitive experiments were designed to obtain a more quantitative evaluation of the mechanism of triplet energy transfer from mod-BSA to the sodium salts of a-naphthylacetic and trans-cinnamic acid. A method for distinguishing between these mechanisms takes advantage of the fact that only the affinity controlled process requires reversible binding of the quencher molecules to the modified-protein before triplet energy transfer occurs. As a result of this we have investigated the inhibitory effect of organic salts, not containing low triplet energy chromophores and possessing easily estimated binding affinities to mod-BSA, on the efficiency of quenching by sodium a-naphthylacetate and transcinnamate. Our earlier efforts (Mariano et al., 1975) had indicated that sodium 3-phenylpropionate and octanoate would ideally serve in this capacity since they do not quench the mod-BSA photoreaction. Several experiments were designed to determine if these salts, which can potentially bind competitively with quenchers to mod-BSA, would inhibit the quenching displayed by cinnamate and a-naphthylacetate. The observed rate of the mod-BSA photoreaction in the presence of varying concentrations of sodium a-naphthylacetate was monitored as a function of sodium octanoate concentration. Indeed, the octanoate salt functioned as an inhibitor of naphthylacetate quenching; the magnitude of the inhibition was dependent on both the octanoate and a-naphthylacetate concentrations. These results are displayed in Fig. 3, which contains plots of the observed mod-BSA
06-
.
[Ql- 4.5 rnM
*[QI=l ImM
'
[Q:= 22mM
0.5
041
I
I
I
0
5
10
I5
I
20
25
3-Phenyi propionate Concentration,
[Il.rnM
Figure 4. Observed mod-BSA photoreaction velocity vs sodium 3-phenylpropionate concentration at fixed sodium trans-cinnamate concentrations ([Q]). The line for [Q] = 0 derives from two independent (W, a) experiments. photoreaction velocity as a function of the octanoate concentration at varying naphthylacetate concentrations. A similar observation was made when the observed photoreaction rates were measured as a function of 3-phenylpropionate and trans-cinnamate concentrations (see Fig. 4). Here again, it appears that the added organic salt, capable of binding to modBSA in competition with the quencher, reduces the quenching effect. Importantly, sodium propionate which is expected to have an extremely low binding affinity to mod-BSA and to only weakly compete with binding of a-naphthylacetate, does not inhibit quenching by a-naphthylacetate (see Fig. 5). DISCUSSION
Our first indications that the affinity controlled mechanism for quenching of the mod-BSA photoreaction might be operative derived from the observations of the relative quenching efficiencies of sodium cl-naphthylacetate, trans-cinnamate, and sorbate and p-naphthyltrimethylammonium chloride. The rather large differences between the quenching efficiencies of these salts, as seen by inspection of Fig. 2 and Table 1, are not easily explainable on the basis of what is known about solution phase triplet energy transfer by the diffusion controlled process. Backstrom and Sandros (1958) and Hammond and co-workers (1963) have shown, independently, that when the triplet energy of a donor is 3-4 kcal/mol higher than that of an acceptor, the rate of energy transfer in solution
[QI=49mM
L 0
157
5
10
15
20
Octanoate concentration.
25 CIl.rnM
Figure 3. Observed mod-BSA photoreaction velocity vs sodium octanoate concentration at fixed sodium a-naphthylacetate concentrations ([Q]).
0
5
10
15
Propionate concentration,
20
I
[I], mM
Figure 5. Observed mod-BSA photoreaction velocity vs sodium propionate concentration at fixed sodium cc-naphthylacetate concentration ( [ Q ] ) .
158
PATRICK S. MARIANO, GEORGE I. GLOVER and TIMOTHY J. WILKINSON
is nearly diffusion controlled, i.e. that the rate constant for quenching (kq) equals that of diffusion (kdiff). When this exchange energy transfer mechanism is adhered to, the quenching efficiencies (@O/@qr where Qio and djq are the reaction quantum yields in the absence and presence of quencher, respectively) for all quenchers of triplet energy below that of the photoreactive substrate are dependent only on the concentration of quencher ([Q]). This relationship is quantified in the familiar Stern-Volmer equation (Stern and Volmer, 1919) (Eq. l), in which T~ is the donor triplet life-time. Small deviations from this behavior have been noted in cases where either back energy transfer
(Sandros, 1964), steric hindrance to orbital overlap between donor and acceptor (Herkstroeter et al., 1966),or repulsion between ionic donors and acceptors of like charge (Cohen et al., 1967) are possible. Our rate data yield a direct measurement of the Q0/Gqratio in terms of the observed relative velocities of the triplet reaction in the absence (V,)and presence (V,) of quencher. The ratio Vo/V,corresponds directly to the quenching efficiency. The results, plotted in Fig. 2 and summarized in Table 1, demonstrate that the quenching efficiencies of the four organic salts, having chromophores of triplet energy below 68 kcal/mol, appear dependent upon factors in addition to quencher concentration. On the basis of these observations, it appears possible that the normal exchange energy transfer mechanism is not solely functioning in quenching of the mod-BSA reaction. A clearer understanding of the quenching data presented above appears possible if consideration is given to another mechanism for energy transfer. Previous investigations by Vaughan and Weber (1970) and Lehrer (1971a and 1971b) have provided evidence for a static quenching mechanism to rationalize observations made on singlet quenching in protein systems. A schematic representation of this process, which we have termed affinity controlled, applied to quenching of the mod-BSA photoreaction is preBSA-ArCOPh
sented in Fig. 6. Accordingly, triplet energy transfer by this mechanism would have its efficiency governed not only by the quencher concentration ([Q]) but also by the mod-BSA association constant (K,) with quencher. A modified Stern-Volmer equation for the affinity controlled energy transfer mechanism, derived in Appendix 1, is given in Eq. 2. An equation similar to this has been derived in a different fashion by Vaughan and Weber (1970). This relationship implies that the quenching efficiency for an affinity controlled process is directly proportional to mod-BSA
association constants for the quencher. Interestingly, Tanford (1972) has shown that the binding sites of native BSA for large organic ligands are anionic specific and that they can be visualized as hydrophobic areas close to centers of positive charge. For example, BSA has a higher affinity for long chain alkyl sulfonates than for trimethylammonium derivatives of the same carbon number. Mod-BSA is expected to display this same binding specificity, since the acetimido-modifications present does not appreciably effect the gross or local charge(s) (Wofsy and Singer, 1963). In light of this, the dramatically different abilities of sodium a-naphthylacetate and P-naphthyltrimethylammonium chloride to quench the protein photoreaction appear to correlate qualitatively with predicted binding affinities of these quenchers to mod-BSA-the anionic acid salt displaying a larger reversible association constant than its cationic counterpart. It is well known (Reynolds et al., 1965; Spector et a!., 1971) that association constants for binding to BSA appear to decrease as the chain length of anionic salts decreases, a possible result of the reduced hydrophobicity of the ligands. Therefore, the comparative quenching efficiencies of sorbate and cinnamate appear to again follow their predicted relative binding constants to the modified protein. The above observations and their interpretation in terms of an affinity controlled mechanism for energy transfer stimulated the design of more definitive experiments. A direct method for demonstrating the
+ a-Naph &
BSA-ArC0Ph.r-Naph
BSA-ArCOPh.a-Naph
2i',:i+ BSA-(ArCOPh)T.r-Naph
BSA-(ArCOPh)*.x-Naph
k, 7 BSA-ArCOPh.(r-Naph)T
BSA-ArCOPh.(cc-Naph)'
decay and+
BSA-ArCOPh
+ x-Naph
dissociation
BSA-ArCOPh BSA-(ArCOPh)T
+ x-Naph
2!.,Fc'BSA-(ArCOPh)T kd,f,
P
BSA-(ArCOPh)'. a-Naph
Figure 6. Schematic for quenching of the mod-BSA photoreaction by the affinity controlled mechanism.
Energy transfer from modified BSA to triplet quenchers
participation of this quenching mode involves measurements of the observed mod-BSA photoreaction velocity in the presence of both the a-naphthylacetate and cinnamate quenchers and other organic salts, which are known to bind to the protein but have no quenching capabilities and can, therefore, serve as inhibitors. Accordingly, if a diffusion controlled mechanism is operative, the velocity of the triplet reaction should be a function of only the quencher concentration and should be independent of the concentration of inhibitors. On the other hand, quenching by the affinity controlled mechanism should be a sensitive function of the competitive inhibitor concentration and its association constant with mod-BSA. The inhibitors would, in effect, reduce the concentration of protein quencher complexes and, thereby, enhance the observed mod-BSA photoreaction velocity by decreasing the quenching efficiency. Lehrer (1971b) has used a similar technique to test for the static quenching mechanism of human serum albumin fluorescence by iodide ion. Data from experiments in which a-naphthylacetate and cinnamate were used as quenchers and octanoate and 3-phenylpropionate, respectively, were employed as inert inhibitors, plotted in Figs. 3 and 4 above, implicate a substantial contribution from the affinity controlled mechanism; both octanoate and 3-phenylpropionate were shown to inhibit quenching. Quantitative evaluation of the data offers additional support for our mechanistic interpretation of these results. A simplified kinetic treatment of the effect of competitive inhibition on the affinity controlled energy transfer mechanism, outlined in Appendix 2, leads to Eq. 3. This equation relates the observed photoreaction velocity (51)and unquenched velocity (V,) to concentrations of inhibitor (I) and quencher (Q) and mod-BSA association constants for quencher (Kp)and inhibitor (Kr). According to this relationship, the function, V,l/Vo-V,I, should be linearly
159
12 11-
10-
COI-22 mM 0
5
10
15
20
3-Phenyl propionate Concentration,
25
[I1 ,mM
Figure 8. Plots of the inhibitor function (&/Vo-VqI) vs sodium 3-phenylpropionate concentration at fixed sodium trans-cinnamate concentrations.
dependent upon inhibitor concentration at fixed quencher concentrations. Interestingly, V,,/Vo-V,j vs [I] plots from several sets of data, employing the a-naphthylacetateloctanoateand cinnamate/3-phenylpropionate combinations are nearly linear (Figs. 7 and 8). As predicted by Eq. 3, both the slopes and intercepts of the lines obtained decrease as the concentration of quencher increases. Another pertinent observation was made when sodium propionate was used as a potential inhibitor of ct-naphthylacetate quenching. The near zero slope of the line obtained by plotting the V,,/V,-V,, ratio vs propionate concentration, dictated by the data shown in Fig. 5, was expected for the affinity controlled mechanism, since the association constant of the modified protein for this salt should be small. This last result enables dismissal of trivial explanations for the observed inhibition of quenching which involve possible interactions between the inhibitors and quenchers. The combined results presented above, although of a preliminary nature, appear to be in agreement with the total or partial operation of an affinity controlled mechanism for energy transfer from mod-BSA triplets to organic salts containing low triplet energy chromophores and high protein binding affinities. Our studies in this area of macromolecule photochemistry are continuing with the aim of obtaining further information on the nature, generality and applications of the observations made.
4-
Acknowledgements-The Robert A. Welch Foundation (Grants A-512 and A-501), the National Science Foundation (Grant No. G.B. 24245) and the donors of the Petroleum Research Fund administered by the American Chemical Society are gratefully acknowledged for their generous financial support of this research. T.J.W. would like to thank the R. A. Welch Foundation for a predoctoral fellowship.
0
5
10
15
20
Octanoate concentration,
25 CI1,mM
Figure 7. Plots of the inhibitor function (V,,/Vo-V,j) VS sodium octanoate concentration at fixed sodium a-naphthylacetate concestrations.
APPENDICES
1. Derivation of mod9ed Stern-Volmer relationship for afinity controlled energy transfer mechanism. The following kinetic sequence for the affinity controlled mechanism for energy transfer from mod-BSA triplets (mod-BSAT) to
PATRICK S. MARIANO, GEORGEI. GLOVER and TIMOTHY J. WILKINSON
I60 quencher (Q)is used.
mod-BSA
I
mod-BSAT
1
=
1
mod-BSA'
A product
mod-BSAT
kd[mod-BSAT]
mod-BSA'
+Q
mod-BSAT.Q k.,,,, Cmod-BSAT] [Q]
mod-BSA
+Q
mod-BSA.Q
mod-BSAT.Q
mod-BSA.Q'
mod-BSA
+ I,
Kt
mod-BSA. I mod-BSAT.I mod-BSAT,l
', ''
Cmd-BSAI [Ql
and V0& is equal to the corresponding quantum yield ratio under simultaneous irradiation conditions. 2. Derivation of relationship between inhibitor concentration and reaction efficiency. The following steps are added to or changed from the kinetic sequence in Appendix 1 in order to account for the presence of competitive inhibitors ( I ) .
KI =
mod-BSAT.I
I=
* product
k,
Division into the quantum yield for the unquenched reaction gives, after simplification, Eq. 2 in which z0 is the intrinsic triplet lifetime, I& + k,),
[mod-BSA. I] [mod-BSA] [I]
KI [I1 1 + K,[I] KO[Q]'I0
+
k,[mod-BSAT. I]
mod-BSA.I
+ K Q[Ql)(k, + kd + k m CQI)
k -2 0k d + k,
K Q = [mod-BSA. Q]
mod-BSA.1
rate constants for triplet decay and reaction, respectively, for all benzophenones, (4) that mod-BSA triplets are long enough lived to enable interaction with quencher to form the mod-BSAT.Q complex with a diffusion controlled rate constant (kd,ff), and (5) that the excited quencher-protein complex undergoes energy transfer (ET) exclusively. The quantum yield (aQ)for mod-BSA reaction in the presence of quencher, using the normal steady state assumption is found to be: (1
k, [mod-BSA']
mod-BSA
The following assumptions are employed: (I) that binding of Q to mod-BSA does not significantly affect its molar extinction coefficient so that the intensities of light absorbed by the free and complexed proteins are controlled by the mod-BSA association constant ( K Q ) for quencher, (2) that intersystem crossing (ISC) of the modBSA triplet is unit efficient, (3) that kd and k, are average
CJQ=
+ KQ[Q]''O
kd[mod-BSAT.I]
The same assumptions made in Appendix 1 are made here along with assumptions that (1) binding of inhibitors does not significantly affect the molar extinction coefficient of mod-BSA so that the relative intensities of light absorbed by free, quenchercomplexed and inhibitor-complexed protein are controlled by the association constants for quencher ( K Q )and inhibitor (Kr), and (2) the average decay (kd) and reaction (k,) rate constants are the same for the free and inhibitor complexed protein triplets. Accordingly, the ratio of the unquenched ( Q 0 ) to quenched (@or) reaction quantum yields in the presence of competitive inhibitors is found to be: -@_O - - I + @QI
(KQCQI+ 1)(1 + kdl~zo[Ql)- 1 1 + Kr (1 + bizso CQI)[I1
Simple algebraic manipulation of this equation gives Eq. 3, in which V,,/VO-V,,is equal to the corresponding quantum yield dividend, C J ~ , / @ ~ - @ ~ ~ .
REFERENCES
Bachstrom, H. L. J., and K. Sandros (1958) Acta Chem. Scand. 12, 823-832. Bregman, J., K. Osaki, G. Schmidt and F. Sonntag (1964) J. Chem. Soc. 2021-2030. Cohen, S. G., R. Thomas and M. Siddiqui (1967) . J . Am. Chem. SOC.89, 584555850, Hammond. G. S., P. A. Leermakers, G. W. Byers and A. A. Lamola (1963) J . Am. Chem. SOC. 85, 267G2671. Herkstroeter, W. G., L. B. Jones and G. S. Hammond (1966) J . Am. Chern. SOC. 88, 4777-4780. Lehrer, S. S . (1971a) Biochemistry 10, 32543263. Lehrer, S. S . (1971b) Biophys. Soc. Abstr. 11, 72a. Mariano, P. S., G. I. Glover and T. J. Wilkinson (1976) Photochem. Photobiol. 23, 147-154.
Energy transfer from modified BSA to triplet quenchers Reynolds, J. A,, S. Herbert, H. Polet and J. Steinhardt (1965) Biochemistry 6 , 937-947. Sandros, K. (1964) Acta Chem. Scand. 18, 2355-2374. Spector, A. A,, J. E. Fletcher and J. D. Ashbrook (1971) Biochemistry 10, 3229-3232. Stern, O., and M. Volmer (1919) Physik. Z . 20, 183188. Tanford, C . (1972) J . Mol. Bid. 67, 59-74. Vaughan, W. M., and G. Weber (1970) Biochemistry 9, 464-473. Wofsy, L., and S . J. Singer (1963) Biochemistry 2, 104-116.
161
Pergamon Press. Printed in Great Britain
THE MECHANISM O F ENERGY TRANSFER FROM POLY-I)-BENZOYLPHENYLACETIMIDO-BOVINE SERUM ALBUMIN TO SMALL-MOLECULE QUENCHERS PATRICK S. MARIANO,*GEORGE I. GLOVER*and TIMOTHY J. WILKINSON Department of Chemistry, Texas A & M University, College Station, TX 77843, USA. (Received 17 July 1975; accepted 16 October 1975)
Abstract-Results of a quantitative photochemical study of poly-p-benzoylphenylacetimido-bovine serum albumin in the presence of small-molecule triplet quenchers are reported. The efficiency of quenching by organic salts containing low triplet energy chromophores is shown to be qualitatively dependent on their predicted association constants to the modified protein. In addition, quenching is inhibited by salts of organic acids which possess high binding affinities for the protein but do not contain chromophores of low triplet energy. Quantitative treatment of the quenching and inhibition data yields results which strongly support the operation of an ‘affinity controlled’ mechanism for triplet energy transfer from the benzophenone moieties of the modified-bovine serum albumin to quenchers such as cc-naphthylacetate and trans-cinnamate.
INTRODUCTION
In the preceding paper (Mariano et al., 1975) we reported an interesting photochemical reaction of poly-p-benzoylphenylacetimido-bovineserum albumin (mod-BSA), a modified protein containing benzophenone moieties covalently linked to the protein backbone through the &-aminogroups of lysine residues. Evidence was presented which indicated that the excited state reactions occurring from the triplet excited benzophenone groups involve the photoreductive addition of the excited carbonyls to carbon-hydrogen bonds within the protein backbone resulting in the introduction of benzhydrol groups as crosslinks in the protein (see Fig. 1 for a schematic representation of the process). Evidence for the nature of the photochemical reaction came from the observation that sodium trans-cinnamate, containing the low triplet energy styryl chromophore (ET = 62 kcal/mol), effectively quenches the mod-BSA photoreaction as a result of its role as an acceptor in the triplet energy transfer process. Support for this view derived from the observation that mod-BSA serves as a photosensitizer of the wellknown (Bregman et al., 1964) cis-trans isomerization of the triplet cinnamic acid salt. Additional evidence to substantiate this conclusion was found using emission spectroscopy. Phosphorescence of the benzophenone chromophores in mod-BSA was efficiently quenched by sodium a-naphthylacetate with concomittant sensitization of naphthalene-like phosphorescence. *The authors to whom correspondence should be communicated. ?The terminology, ‘affinity controlled’, is suggested by the fact that energy transfer by this mechanism will have its efficiency controlled by the affinity of the protein sensitizer for the quencher.
Our studies of the mod-BSA photoreaction have led to further investigations designed to determine if an affinity controlledt (static) (Vaughan and Weber, 1970; Lehrer, 1971a, 1971b) or diffusion controlled triplet energy transfer mechanism is operating in those cases where quenching of the mod-BSA photoreaction was detected. In the former mechanism, energy transfer from the triplet excited benzophenone moieties occurs to quencher molecules while they are reversibly bound to the modified protein. We would like to report the results of this investigation, which strongly implicate the operation of an affinity controlled mechanism for triplet energy transfer from mod-BSA to quenchers such as the sodium salts of a-naphthylacetic and trans-cinnamic acids.
Figure 1. Schematic representation of the mod-BSA photoreaction which involves photoreductive addition of the benzophenone moieties to a-CH bonds within the protein backbone. MATERIALS AND METHODS
General. Poly-p-benzoylphenylacetimido-bovineserum albumin was prepared according to procedure reported earlier (Mariano et al., 1975). trans-Cinnamic acid (Sigma) was twice recrystallized prior to use. /3-Naphthyltrimethylammonium chloride was prepared from the corresponding iodide by anion exchange chromatography. All other compounds were commercially available and were of reagent grade or better. All,buffered solutions were made with the same pH 8.0 buffer consisting of 0.05M KH2P04 and 0.1 M KCI, adjusted to pH 8.0 with 5 N KOH. UV spectral analyses were accomplished using a Beckman Acta 111 UVvisible spectrophotometer.All simultaneous irradiations of
155
PATRICK S. MARIANO,GEORGE I. GLOVER and TIMOTHY J. WILKINSON
156
65-
Quencher concentration.
[QI,
mM
Figure 2. Plots of mod-BSA photoreaction quenching efficiencies (Vo/V,) vs quencher (Q) concentration for sodium r-naphthylacetate (@), trans-cinnamate (W), sorbate (W), octanoate (0)and 3-phenylpropionate (U), and trimethylP-naphthylammonium chloride (V)as quenchers. multiple samples were carried out using an apparatus consisting of a merry-go-round at the center of which was located a 450W Hanovia medium pressure lamp surrounded by a uranium glass filter (T330nm = 5%, &sonrn = 45%, T370n,,, = 76%) in a water-cooled quartz immersion well. All photolysis solutions were degassed in uacuo and saturated with N, prior to use. However, we have found that dissolved oxygen has no effect upon the rate of the modified-protein photoreaction. Irradiations of mod-BSA in the presence of’ varying concentrations of potential quenchers. Stock solutions of the following potential quenchers were prepared in pH 8 buffer: sodium a-naphthylacetate (59 mM), j-naphthyltrimethylammonium chloride (59 mM), sodium trans-cinnamate (59 mM), sodium sorbate (59 mM), sodium octanoate (59 mM) and sodium 3-phenylpropionate (59 mM). Aliquots of these solutions were diluted with appropriate amounts of buffer to give a series of solutions of varying concentrations. One md portions of these solutions were mixed with 2 md of a mod-BSA solution (65 p M in pH 8 buffer) to give final solutions containing the varying concentrations of the four quenchers as indicated in Fig. 2. These mod-BSA solutions along with a control consisting of mod-BSA alone were simultaneously irradiated for 2 h in the merry-go-round apparatus using uranium glass-filtered light. One m t of each photolysate was gel filtered on Sephadex G-25 (fine) to separate quenchers from protein. The protein-containing peaks were collected and diluted to 10.0me with pH 8 buffer. The absorbances at 260 nm of these solutions were used to obtain the relative observed mod-BSA reaction velocities according to the following method. During an independent and extensive irradiation of mod-BSA (7.2 pM), the absorbance at 260 nm decreased from its initial value of 0.988 (A,) and after ca.
6 h leveled at a value of 0.490 ( A z ) . A plot of the natural log of the mole fraction of benzophenone chromophores remaining at time 2, expressed as AO-A/Ao-A,, where A is the absorbance at t, vs time is linear throughout the duration of the irradiation. As a result, the absorbances of proteincontaining solutions, from the 2 h simultaneous irradiations in the presence of quenchers and after chromatographic separation, are inversely proportional to the observed mod-BSA reaction velocities. The results of these experiments are recorded in Fig. 2. Irradiation of mod-BSA in the presence of quenchers and inhibitors. Sodium a-naphthylacetatr and octanoate. Thirtytwo 3 mY solutions each containing mod-BSA (42.6 p M ) in sets of eight containing sodium a-naphthyacetate at concentrations of 0.0, 14.9, 37.0, and 74.1 mM were prepared. Each set of eight solutions of constant a-naphthylacetate concentration contained sodium octanoate in the following concentrations: 0.0, 2.46, 4.91, 7.36, 11.1, 14.8, 19.6 and 24.5 mM. The solvent in each case was pH 8 buffer. These were simultaneously irradiated in a merry-go-round apparatus with uranium glass-filtered light for 3 h. The photolysates were individually gel-filtered on Sephadex G-25 (fine); the protein-containing peaks were collected and diluted to a fixed volume and analyzed by ultraviolet spectroscopy to determine the observed mod-BSA photoreaction velocities. The results from this experiment in terms of relative observed reaction velocities for the mod-BSA are plotted in Fig. 3. Sodium trans-cinnamate and 3-phenylpropionate. Thirtytwo 3 mf solutions containing mod-BSA (42.6 p M ) , sodium trans-cinnamate (0.0, 13.2, 32.9, and 65.9 mM) and sodium 3-phenylpropionate (0.0, 24.6,4.91, 7.36, 11.1, 14.8, 19.6 and 24,5mM) prepared in the same way as described above in pH 8 buffer were simultaneously irradiated in a merrygo-round apparatus using uranium glass-filtered light for 3 h. Analyses of the extent and relative observed rate of the mod-BSA reaction were conducted in the same mannei as described above. Results of this experiment are recorded in Fig. 4. Sodium a-naphthylacetate and propionate. Thirty-two 3 m/ samples containing mod-BSA and sodium a-naphthylacetate, in the same concentrations as for the a-naphthylacetate/octanoate experiment and sodium propionate (0.0, 2.46. 4.91, 7.36, 11.1, 14.8, 19.6 and 24.5mM) were simultaneously irradiated and analyzed as described above. Results from this experiment are recorded in Fig. 5. RESULTS
Quenching of’ the mod-BSA photoreaction using a variety oforganic salts. In an attempt to first qualitatively evaluate the mechanism of triplet energy transfer from mod-BSA, excited at wavelengths greater than 320nm, an exploratory investigation of the effect of quenchers on the mod-BSA photoreaction was conducted. In Fig. 2 and Table 1 are summarized the
Table 1. Mod-BSA photoreaction quenching efficiencies for various low triplet energy quenchers
*
Quenchers
Triplet Energies (kcal/mol)
Relative Quenching Efficiency ?.
Sodium a-Napthylacetate
61.0
8.57
Sodium =-Cinnamate
61.5
3.77
Sodium Sorbate
59.5
1.15
Trimthy 1-B-naphthylammonium chloride
61.0
1.00
*Assumed on basis of chromophores present in salts. ?Based on the slopes of the Vo/V, vs [Q] plots, and setting that for the ammonium salt as 1.
Energy transfer from modified BSA to triplet quenchers results of these experiments in which the observed velocity of the mod-BSA photochemical reaction was measured as a function of concentrations of various organic salts. The sodium salts of a-naphthylacetic, trans-cinnamic, sorbic, octanoic, and 3-phenyl-propionic acids, and b-naphthyItrimethylammonium chloride were chosen on the basis of either their potential binding affinities to mod-BSA and/or their possession of low triplet-energy chromophores. Plotted in Fig. 2 are the ratios of the observed photochemical reaction velocities in the presence and absence of these compounds (Vo/V,)vs their concentrations,
CQJ Quenching of the mod-BSA photoreaction in the presence of competitive inhibitors. Definitive experiments were designed to obtain a more quantitative evaluation of the mechanism of triplet energy transfer from mod-BSA to the sodium salts of a-naphthylacetic and trans-cinnamic acid. A method for distinguishing between these mechanisms takes advantage of the fact that only the affinity controlled process requires reversible binding of the quencher molecules to the modified-protein before triplet energy transfer occurs. As a result of this we have investigated the inhibitory effect of organic salts, not containing low triplet energy chromophores and possessing easily estimated binding affinities to mod-BSA, on the efficiency of quenching by sodium a-naphthylacetate and transcinnamate. Our earlier efforts (Mariano et al., 1975) had indicated that sodium 3-phenylpropionate and octanoate would ideally serve in this capacity since they do not quench the mod-BSA photoreaction. Several experiments were designed to determine if these salts, which can potentially bind competitively with quenchers to mod-BSA, would inhibit the quenching displayed by cinnamate and a-naphthylacetate. The observed rate of the mod-BSA photoreaction in the presence of varying concentrations of sodium a-naphthylacetate was monitored as a function of sodium octanoate concentration. Indeed, the octanoate salt functioned as an inhibitor of naphthylacetate quenching; the magnitude of the inhibition was dependent on both the octanoate and a-naphthylacetate concentrations. These results are displayed in Fig. 3, which contains plots of the observed mod-BSA
06-
.
[Ql- 4.5 rnM
*[QI=l ImM
'
[Q:= 22mM
0.5
041
I
I
I
0
5
10
I5
I
20
25
3-Phenyi propionate Concentration,
[Il.rnM
Figure 4. Observed mod-BSA photoreaction velocity vs sodium 3-phenylpropionate concentration at fixed sodium trans-cinnamate concentrations ([Q]). The line for [Q] = 0 derives from two independent (W, a) experiments. photoreaction velocity as a function of the octanoate concentration at varying naphthylacetate concentrations. A similar observation was made when the observed photoreaction rates were measured as a function of 3-phenylpropionate and trans-cinnamate concentrations (see Fig. 4). Here again, it appears that the added organic salt, capable of binding to modBSA in competition with the quencher, reduces the quenching effect. Importantly, sodium propionate which is expected to have an extremely low binding affinity to mod-BSA and to only weakly compete with binding of a-naphthylacetate, does not inhibit quenching by a-naphthylacetate (see Fig. 5). DISCUSSION
Our first indications that the affinity controlled mechanism for quenching of the mod-BSA photoreaction might be operative derived from the observations of the relative quenching efficiencies of sodium cl-naphthylacetate, trans-cinnamate, and sorbate and p-naphthyltrimethylammonium chloride. The rather large differences between the quenching efficiencies of these salts, as seen by inspection of Fig. 2 and Table 1, are not easily explainable on the basis of what is known about solution phase triplet energy transfer by the diffusion controlled process. Backstrom and Sandros (1958) and Hammond and co-workers (1963) have shown, independently, that when the triplet energy of a donor is 3-4 kcal/mol higher than that of an acceptor, the rate of energy transfer in solution
[QI=49mM
L 0
157
5
10
15
20
Octanoate concentration.
25 CIl.rnM
Figure 3. Observed mod-BSA photoreaction velocity vs sodium octanoate concentration at fixed sodium a-naphthylacetate concentrations ([Q]).
0
5
10
15
Propionate concentration,
20
I
[I], mM
Figure 5. Observed mod-BSA photoreaction velocity vs sodium propionate concentration at fixed sodium cc-naphthylacetate concentration ( [ Q ] ) .
158
PATRICK S. MARIANO, GEORGE I. GLOVER and TIMOTHY J. WILKINSON
is nearly diffusion controlled, i.e. that the rate constant for quenching (kq) equals that of diffusion (kdiff). When this exchange energy transfer mechanism is adhered to, the quenching efficiencies (@O/@qr where Qio and djq are the reaction quantum yields in the absence and presence of quencher, respectively) for all quenchers of triplet energy below that of the photoreactive substrate are dependent only on the concentration of quencher ([Q]). This relationship is quantified in the familiar Stern-Volmer equation (Stern and Volmer, 1919) (Eq. l), in which T~ is the donor triplet life-time. Small deviations from this behavior have been noted in cases where either back energy transfer
(Sandros, 1964), steric hindrance to orbital overlap between donor and acceptor (Herkstroeter et al., 1966),or repulsion between ionic donors and acceptors of like charge (Cohen et al., 1967) are possible. Our rate data yield a direct measurement of the Q0/Gqratio in terms of the observed relative velocities of the triplet reaction in the absence (V,)and presence (V,) of quencher. The ratio Vo/V,corresponds directly to the quenching efficiency. The results, plotted in Fig. 2 and summarized in Table 1, demonstrate that the quenching efficiencies of the four organic salts, having chromophores of triplet energy below 68 kcal/mol, appear dependent upon factors in addition to quencher concentration. On the basis of these observations, it appears possible that the normal exchange energy transfer mechanism is not solely functioning in quenching of the mod-BSA reaction. A clearer understanding of the quenching data presented above appears possible if consideration is given to another mechanism for energy transfer. Previous investigations by Vaughan and Weber (1970) and Lehrer (1971a and 1971b) have provided evidence for a static quenching mechanism to rationalize observations made on singlet quenching in protein systems. A schematic representation of this process, which we have termed affinity controlled, applied to quenching of the mod-BSA photoreaction is preBSA-ArCOPh
sented in Fig. 6. Accordingly, triplet energy transfer by this mechanism would have its efficiency governed not only by the quencher concentration ([Q]) but also by the mod-BSA association constant (K,) with quencher. A modified Stern-Volmer equation for the affinity controlled energy transfer mechanism, derived in Appendix 1, is given in Eq. 2. An equation similar to this has been derived in a different fashion by Vaughan and Weber (1970). This relationship implies that the quenching efficiency for an affinity controlled process is directly proportional to mod-BSA
association constants for the quencher. Interestingly, Tanford (1972) has shown that the binding sites of native BSA for large organic ligands are anionic specific and that they can be visualized as hydrophobic areas close to centers of positive charge. For example, BSA has a higher affinity for long chain alkyl sulfonates than for trimethylammonium derivatives of the same carbon number. Mod-BSA is expected to display this same binding specificity, since the acetimido-modifications present does not appreciably effect the gross or local charge(s) (Wofsy and Singer, 1963). In light of this, the dramatically different abilities of sodium a-naphthylacetate and P-naphthyltrimethylammonium chloride to quench the protein photoreaction appear to correlate qualitatively with predicted binding affinities of these quenchers to mod-BSA-the anionic acid salt displaying a larger reversible association constant than its cationic counterpart. It is well known (Reynolds et al., 1965; Spector et a!., 1971) that association constants for binding to BSA appear to decrease as the chain length of anionic salts decreases, a possible result of the reduced hydrophobicity of the ligands. Therefore, the comparative quenching efficiencies of sorbate and cinnamate appear to again follow their predicted relative binding constants to the modified protein. The above observations and their interpretation in terms of an affinity controlled mechanism for energy transfer stimulated the design of more definitive experiments. A direct method for demonstrating the
+ a-Naph &
BSA-ArC0Ph.r-Naph
BSA-ArCOPh.a-Naph
2i',:i+ BSA-(ArCOPh)T.r-Naph
BSA-(ArCOPh)*.x-Naph
k, 7 BSA-ArCOPh.(r-Naph)T
BSA-ArCOPh.(cc-Naph)'
decay and+
BSA-ArCOPh
+ x-Naph
dissociation
BSA-ArCOPh BSA-(ArCOPh)T
+ x-Naph
2!.,Fc'BSA-(ArCOPh)T kd,f,
P
BSA-(ArCOPh)'. a-Naph
Figure 6. Schematic for quenching of the mod-BSA photoreaction by the affinity controlled mechanism.
Energy transfer from modified BSA to triplet quenchers
participation of this quenching mode involves measurements of the observed mod-BSA photoreaction velocity in the presence of both the a-naphthylacetate and cinnamate quenchers and other organic salts, which are known to bind to the protein but have no quenching capabilities and can, therefore, serve as inhibitors. Accordingly, if a diffusion controlled mechanism is operative, the velocity of the triplet reaction should be a function of only the quencher concentration and should be independent of the concentration of inhibitors. On the other hand, quenching by the affinity controlled mechanism should be a sensitive function of the competitive inhibitor concentration and its association constant with mod-BSA. The inhibitors would, in effect, reduce the concentration of protein quencher complexes and, thereby, enhance the observed mod-BSA photoreaction velocity by decreasing the quenching efficiency. Lehrer (1971b) has used a similar technique to test for the static quenching mechanism of human serum albumin fluorescence by iodide ion. Data from experiments in which a-naphthylacetate and cinnamate were used as quenchers and octanoate and 3-phenylpropionate, respectively, were employed as inert inhibitors, plotted in Figs. 3 and 4 above, implicate a substantial contribution from the affinity controlled mechanism; both octanoate and 3-phenylpropionate were shown to inhibit quenching. Quantitative evaluation of the data offers additional support for our mechanistic interpretation of these results. A simplified kinetic treatment of the effect of competitive inhibition on the affinity controlled energy transfer mechanism, outlined in Appendix 2, leads to Eq. 3. This equation relates the observed photoreaction velocity (51)and unquenched velocity (V,) to concentrations of inhibitor (I) and quencher (Q) and mod-BSA association constants for quencher (Kp)and inhibitor (Kr). According to this relationship, the function, V,l/Vo-V,I, should be linearly
159
12 11-
10-
COI-22 mM 0
5
10
15
20
3-Phenyl propionate Concentration,
25
[I1 ,mM
Figure 8. Plots of the inhibitor function (&/Vo-VqI) vs sodium 3-phenylpropionate concentration at fixed sodium trans-cinnamate concentrations.
dependent upon inhibitor concentration at fixed quencher concentrations. Interestingly, V,,/Vo-V,j vs [I] plots from several sets of data, employing the a-naphthylacetateloctanoateand cinnamate/3-phenylpropionate combinations are nearly linear (Figs. 7 and 8). As predicted by Eq. 3, both the slopes and intercepts of the lines obtained decrease as the concentration of quencher increases. Another pertinent observation was made when sodium propionate was used as a potential inhibitor of ct-naphthylacetate quenching. The near zero slope of the line obtained by plotting the V,,/V,-V,, ratio vs propionate concentration, dictated by the data shown in Fig. 5, was expected for the affinity controlled mechanism, since the association constant of the modified protein for this salt should be small. This last result enables dismissal of trivial explanations for the observed inhibition of quenching which involve possible interactions between the inhibitors and quenchers. The combined results presented above, although of a preliminary nature, appear to be in agreement with the total or partial operation of an affinity controlled mechanism for energy transfer from mod-BSA triplets to organic salts containing low triplet energy chromophores and high protein binding affinities. Our studies in this area of macromolecule photochemistry are continuing with the aim of obtaining further information on the nature, generality and applications of the observations made.
4-
Acknowledgements-The Robert A. Welch Foundation (Grants A-512 and A-501), the National Science Foundation (Grant No. G.B. 24245) and the donors of the Petroleum Research Fund administered by the American Chemical Society are gratefully acknowledged for their generous financial support of this research. T.J.W. would like to thank the R. A. Welch Foundation for a predoctoral fellowship.
0
5
10
15
20
Octanoate concentration,
25 CI1,mM
Figure 7. Plots of the inhibitor function (V,,/Vo-V,j) VS sodium octanoate concentration at fixed sodium a-naphthylacetate concestrations.
APPENDICES
1. Derivation of mod9ed Stern-Volmer relationship for afinity controlled energy transfer mechanism. The following kinetic sequence for the affinity controlled mechanism for energy transfer from mod-BSA triplets (mod-BSAT) to
PATRICK S. MARIANO, GEORGEI. GLOVER and TIMOTHY J. WILKINSON
I60 quencher (Q)is used.
mod-BSA
I
mod-BSAT
1
=
1
mod-BSA'
A product
mod-BSAT
kd[mod-BSAT]
mod-BSA'
+Q
mod-BSAT.Q k.,,,, Cmod-BSAT] [Q]
mod-BSA
+Q
mod-BSA.Q
mod-BSAT.Q
mod-BSA.Q'
mod-BSA
+ I,
Kt
mod-BSA. I mod-BSAT.I mod-BSAT,l
', ''
Cmd-BSAI [Ql
and V0& is equal to the corresponding quantum yield ratio under simultaneous irradiation conditions. 2. Derivation of relationship between inhibitor concentration and reaction efficiency. The following steps are added to or changed from the kinetic sequence in Appendix 1 in order to account for the presence of competitive inhibitors ( I ) .
KI =
mod-BSAT.I
I=
* product
k,
Division into the quantum yield for the unquenched reaction gives, after simplification, Eq. 2 in which z0 is the intrinsic triplet lifetime, I& + k,),
[mod-BSA. I] [mod-BSA] [I]
KI [I1 1 + K,[I] KO[Q]'I0
+
k,[mod-BSAT. I]
mod-BSA.I
+ K Q[Ql)(k, + kd + k m CQI)
k -2 0k d + k,
K Q = [mod-BSA. Q]
mod-BSA.1
rate constants for triplet decay and reaction, respectively, for all benzophenones, (4) that mod-BSA triplets are long enough lived to enable interaction with quencher to form the mod-BSAT.Q complex with a diffusion controlled rate constant (kd,ff), and (5) that the excited quencher-protein complex undergoes energy transfer (ET) exclusively. The quantum yield (aQ)for mod-BSA reaction in the presence of quencher, using the normal steady state assumption is found to be: (1
k, [mod-BSA']
mod-BSA
The following assumptions are employed: (I) that binding of Q to mod-BSA does not significantly affect its molar extinction coefficient so that the intensities of light absorbed by the free and complexed proteins are controlled by the mod-BSA association constant ( K Q ) for quencher, (2) that intersystem crossing (ISC) of the modBSA triplet is unit efficient, (3) that kd and k, are average
CJQ=
+ KQ[Q]''O
kd[mod-BSAT.I]
The same assumptions made in Appendix 1 are made here along with assumptions that (1) binding of inhibitors does not significantly affect the molar extinction coefficient of mod-BSA so that the relative intensities of light absorbed by free, quenchercomplexed and inhibitor-complexed protein are controlled by the association constants for quencher ( K Q )and inhibitor (Kr), and (2) the average decay (kd) and reaction (k,) rate constants are the same for the free and inhibitor complexed protein triplets. Accordingly, the ratio of the unquenched ( Q 0 ) to quenched (@or) reaction quantum yields in the presence of competitive inhibitors is found to be: -@_O - - I + @QI
(KQCQI+ 1)(1 + kdl~zo[Ql)- 1 1 + Kr (1 + bizso CQI)[I1
Simple algebraic manipulation of this equation gives Eq. 3, in which V,,/VO-V,,is equal to the corresponding quantum yield dividend, C J ~ , / @ ~ - @ ~ ~ .
REFERENCES
Bachstrom, H. L. J., and K. Sandros (1958) Acta Chem. Scand. 12, 823-832. Bregman, J., K. Osaki, G. Schmidt and F. Sonntag (1964) J. Chem. Soc. 2021-2030. Cohen, S. G., R. Thomas and M. Siddiqui (1967) . J . Am. Chem. SOC.89, 584555850, Hammond. G. S., P. A. Leermakers, G. W. Byers and A. A. Lamola (1963) J . Am. Chem. SOC. 85, 267G2671. Herkstroeter, W. G., L. B. Jones and G. S. Hammond (1966) J . Am. Chern. SOC. 88, 4777-4780. Lehrer, S. S . (1971a) Biochemistry 10, 32543263. Lehrer, S. S . (1971b) Biophys. Soc. Abstr. 11, 72a. Mariano, P. S., G. I. Glover and T. J. Wilkinson (1976) Photochem. Photobiol. 23, 147-154.
Energy transfer from modified BSA to triplet quenchers Reynolds, J. A,, S. Herbert, H. Polet and J. Steinhardt (1965) Biochemistry 6 , 937-947. Sandros, K. (1964) Acta Chem. Scand. 18, 2355-2374. Spector, A. A,, J. E. Fletcher and J. D. Ashbrook (1971) Biochemistry 10, 3229-3232. Stern, O., and M. Volmer (1919) Physik. Z . 20, 183188. Tanford, C . (1972) J . Mol. Bid. 67, 59-74. Vaughan, W. M., and G. Weber (1970) Biochemistry 9, 464-473. Wofsy, L., and S . J. Singer (1963) Biochemistry 2, 104-116.
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