Proc. Nat. Acad. Sci. USAVol. 71, No. 5, pp. 1686-1690, May 1974

The Activity-Related Ionization in Carbonic Anhydrase(metalloenzymes/enzyme mechanism/imidazole chelates)

DAVID W. APPLETON AND BIBUDHENDRA SARKAR

The Division of Biochemistry, The Research Institute, The Hospital for Sick Children, Toronto; and the Department of Biochemistry,University of Toronto, Toronto, Ontario, Canada

Communicated by Jacob Bigeleisen, January 25, 1974

ABSTRACT The catalytic activity of carbonic anhy-drase (EC 4.2.1.1) is linked to the ionization of a group inclose proximity to the essential zinc ion. Studies have beenundertaken to delineate the ionizations germane to theactive-site chelate system. Several imidazole ligand sys-tems were studied in order to approach a representativechelate. The simplest involved the complexation of Zn(II)by imidazole and by N-methylimidazole. As well, two bi-dentate systems, Zn(II)-4,4'-bis-imidazoylmethane andCo(II)-cyclic-L-histidyl-L-histidine were investigated. Itwas found that in a species containing metal-bound waterand imidazole coordinated by means of the pyridiniumnitrogen, the most acidic group was the pyrroleN-H in the imidazole ring. By the use of N-methylimid-azole, the pKa of a metal-bound water molecule in a tri-imidazole ligand field was found to be 9.1. Noting thepreference for labilization of the pyrrole hydrogen, thecatalytic features of carbonic anhydrase are reexaminedassuming that the pK... is associated with the N-H ioniza-tion, and not with the ionization of metal-bound water.

In 1964, Kernohan established a pH dependence for bovinecarbonic anhydrase (carbonate hydro-lyase; EC 4.2.1.1)characterized by a pKenz of 6.9 (1). Subsequently, kineticinvestigations were extended to include the human B and Cisoenzymes (2, 3). The high-activity human C enzyme wasfound to have a pH dependence similar to that described forthe bovine enzyme. Further work has revealed that the humanB enzyme has a lower turnover number coupled with a morealkaline pH dependence, having a pKenz value near 8.2 (4, 5).

Although the pH dependence of the intrinsic activity hasbeen known for quite some time, the nature of the ionizinggroup responsible for the sigmoidal behavior has been, andstill is, the seat of much discussion. It has been well estab-lished that the ionizing group must be intimately associatedwith the essential metal ion cofactor (6). Changes in the co-valent domain of the metal ion can be selectively monitoredif the native zinc is replaced by cobalt (7). Visible spectral(8, 9) and related analyses (10, 11) of the substituted enzymereveal a close parallelism between the development of in-trinsic activity and profound changes in the ligand system.The interaction of 83C1 with the bovine enzyme indicates thatenzymic inhibition is concurrent with the binding of one ionper enzyme molecule, directly to the metal ion. This anionicinhibition is intimately associated with the pH dependence,

as a plot of the apparent pKenz against [Cl-] yields the ex-pected pKenz when extrapolated to zero chloride concentra-tion (12).Assuming that the ionization is metal ion linked, there

exist two possible interpretations. Either the dissociationoriginates in the preexisting ligand system, or the ligandsare exchanged, inducing the ionization of the incoming ligand.Of the two, the latter choice appears less attractive, for thefollowing reason. It has been found from x-ray crystallo-graphic studies on the human C isoenzyme, crystallized atpH 8.5, that the zinc ion is tetrahedrally coordinated to threeimidazole rings and most likely a solvent molecule (13). Frommagnetic moment (14), spectral (15), and magnetic circulardichroism results (16), this situation is thought to be main-tained in solution. Taken most simply then, the x-ray studydefines the ligand field of the active enzyme. Presumably,a ligand exchange leading to the active enzyme would alreadyhave occurred. But, Coleman (4), by cyanide titration, hasfound the ionizing group to have an intrinsic pKa> 11, a pKadifficult to attribute to the ionization of a protonated imidazoleside chain. Thus, in light of the arguments that would beneeded to rationalize a ligand substitution process, the alterna-tive of an ionization in the preexisting metal ion-ligand sys-tem seems more straightforward.

In the alternative hypothesis, the dissociable hydrogens arethe histidyl pyrrole hydrogens* and the metal-bound watermolecule protons. The pyrrole hydrogens have as yet not beendiscussed in connection with the ionization, presumably be-cause of their intrinsic pK.; the pKa of neutral imidazoleis 14.5 (17). The known versatility of coordinated hydroxide(18, 19) coupled with its compatibility with most of the avail-able criteria has led many workers (6, 20, 21) to accept thewater dissociation hypothesis. Unfortunately, due to theproblems inherent in the experimental design, little directevidence is available to substantiate or refute this hypothesis.As well, Koenig and Brown (22) have questioned the proposal,being unable to reconcile either the nuclear magnetic relaxa-tion dispersion data or Ward's 35CI relaxation data with theionization of a metal-bound water molecule.

In an attempt to more fully correlate the observed param-eters pertinent to this ionization, we have begun an investi-

Abbreviations: Complexes of the type MpHqAr are defined asfollows: p, q, r are the respective stoichiometries of the metal ionM, the hydrogen ion H, and the uncharged ligand A. The sub-script, -q, indicates the uptake by the complex of q additionalequivalents of base, due either to the titration of pyrrole or com-plexed water protons.

* Imidazoles are amphoteric compounds. Due to the basicity ofthe "pyridine-type" nitrogen, imidazoles readily form salts withacids and complexes with most metal ions. Imidazoles with the"pyrrole-type" N-H intact are weak acids with pKa values onthe order of 14.

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Activity-Related Ionization in Carbonic Anhydrase 1687

gation into the dissociations associated with mixed complexesof the type metal ion [Zn(II) or Co(II)I, imidazole, water.In doing so, we have hopefully provided a case for an evalua-tion of the possibility of pyrrole hydrogen ionization, as wellas some evidence about the possible pH dependencies for thetwo relevant ionizations.

MATERIALS AND METHODAnalytical grade imidazole was twice recrystallized frombenzene. The N-methylimidazole obtained from the AldrichChemical Co. was distilled under reduced pressure and storedunder dry nitrogen. The cycdic-ihistidyl-ihistidine wasprepared according to the method of Fischer and Suzuki(23), and twice recrystallized under argon, from hot water.The 4,4'-bis-imidazoylmethane was isolated as the dinitratesalt by a synthesis modified from Drey and Fruton (24).Ligand stock solution concentrations were checked repeatedlyby titration with standardized KOH.

Stock solutions of zinc nitrate were prepared by dissolvinganalytical grade zinc oxide in redistilled nitric acid. The co-balt nitrate solutions were prepared from analytical gradeCo(NO3)2-6H20. The metal stock solutions were standard-ized by EDTA titration with xylenol orange as indicator.

All titrations were carried out at 250 4 0.10 under washedargon with 0.16 M KNO3 as background electrolyte. Theelectrode linearity was calibrated with four National Bureauof Standards primary pH standard solutions, pH values 1.650,4.008, 7.413, and 9.180 (25).

All systems excepting the Zn(II)-N-methylimidazole werestudied by continuous titration, with the equipment andmethod described in ref. 26. To avoid the problem of metalhydroxide precipitation, the Zn(II)-N-methylimidazole sys-tem was studied by discontinuous titration, with a medialligand metal ratio of 258/1. At each of 30 pH values between3.898 and 11.005, a series of five separate potentiostatic titra-tions was performed by the sequential addition of 0.1 through0.5 ml of a 60.00 mM Zn(II) stock solution to 50.0 ml of an77.37 mM solution of the ligand. The titration results wereanalyzed with the aid of our programs, PLOT, GUESS, andLEASK (27), on a GE-440 computer.

Infrared spectra were recorded on a Beckman IR-20A.Visible spectra were recorded at 250 on a Cary 15 recordingspectrophotometer. The titration apparatus was connected toa sealed cuvette, of path length 5.0 cm, by a syringe-drivenflow system.

RESULTSTitration, with aqueous base of a solution 1.0 mM with respectto Zn(II) and 10.0 mM with respect to imidazole, causes theprecipitation, beginning about pH 7.5, of a flocculent whitesolid. X-ray diffraction studies have shown that the precipi-tate, zinc imidazolate, is an infinite polymer in which eachmetal ion is tetrahedrally coordinated to four ligand mole-cules, with each imidazole acting as a bidentate link betweentwo metal ions (28). The loss of the pyrrole hydrogen has beenverified by infrared studies that demonstrated the absenceof N-H stretching bands in the solid (29). We have completelysubstantiated the above findings by stoichiometric analysisand by infrared studies on precipitate isolated below pH8 from both H20 and D20 solutions.

Titration studies of the zinc imidazole system have, to date,not taken into account the pyrrole ionization (30), although

_ 02cNae

5.5 6.0 6.5 7.0 7.5pH

FIG. 1. The partial species distribution for a solution of 1.15mM Zn(II), 19.64 mM imidazole, and 0.16 M KNO, at 250. Thespecies shown are: (1) MA; (2) MA2; (3) MH-1A2; (4) MH-2A2.Values on the ordinate have been multiplied by 10-2.

the presence of the precipitate is proof that the species(MH-2A2)n is forming. This factor would then introduce aheavy bias into ni calculations of the initial stepwise constants.As a consequence, studies were undertaken to reevaluatethe system, both to better understand the stepwise com-plexation and to describe the pH dependence of the pyrroledeprotonation. In short, complete analysis proved. impos-sible. The amount of any species present in the system is ob-tained by a least-squares fit (Program LEASK) of the speciescurve shapes (Program GUESS) to the calculated boundmetal function. However, it was found that five stepwisespecies, plus the pyrrole species, occur simultaneously withinone pH unit. Given normal fitting errors and the lack of databeyond pH 7.5, there was no unique solution. In Fig. 1 areshown representative results. In curve 1, the species MA isrigorously fitted and log #lo, = 2.48 agrees well with pub-lished values (30).The species MA2, curve 2, is inordinately broad. Its pro-

portion depends heavily on the amount of MA3 present in thesystem. But MA3 and MA4 cannot be calculated separatelybecause the curve shapes are prematurely truncated due tothe precipitation problem. The lower limit of MA2 has beenincluded for interest, and the composite MA3 and MA4 hasbeen omitted. Since none of the pyrrole species peak beforeprecipitation, rigorous fittings in the context of this methodis negated. However, information on the pH dependence canbe obtained since the "pyrrole deprotonation envelope" canbe calculated. We have fitted the envelope, assuming twospecies MH-1A2 and MH-2A2. Obviously the second specieswill not exist to any great extent, in nonpolynuclear form.However, all polymers of either species will have maxima atlower respective pH values, such that the fitted curves rep-resent the most alkaline pH dependencies.

In an effort to extend the analysis of the pyrrole ionizationto a broader pH range, two bidentate ligands were subse-quently studied. Drey and Fruton (31), reported that 4,4'-bis-imidazoylmethane readily forms a bis complex, MA2,with zinc. At the time, they reported that further ionizationof an unspecified nature did occur. Reanalysis of this systemshowed that the species MA2 declined with increasing pHconcomitant with the dissociation of pyrrole hydrogens andsubsequent precipitation. The first ionization fits as MH-1A2and again because of precipitation, species leading fromMH_-A2 to (MH-2A2). were fitted under MH-2A2. The pKa

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0.50N

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0pH

FIG. 2. The species distribution for a solution of 0.299 mMZn(H), 77.0 mM N-methylimidazole, and 0.16 M KNOs at 250.The species shown are: (I) unbound metal; (2) MA; (8) MA2;(4) MA3; (5) MAX; (6) MA.5; (7) MA6; (8) MH_ IA4; (9) MH-rA4.

Of the pyrrole ionization giving rise to MH-jA2 is 7.90. Thisis to be compared with a value of 7.0-7.3 for MH-,A2 in theZn(HI)-imidazole system (Table 1).

It has been reported that in cyclic-L-histidyl-L-histidine,the two imidazole side chains share, in close proximity, thespace over the diketopiperazine ring (32). To exploit theobvious advantages such a bidentate ligand may possess,an analysis of the interaction with Co(HI) was carried out.Cyclic-L-histidyl-L-histidine forms a single complex withCo(II), MA (log #lo, = 2.68). Above pH 7, MA begins dis-sociating pyrrole hydrogens. The dissociation of a secondpyrrole hydrogen from MA leads to polynucleation althoughno precipitate is seen until above pH 9. The species MH-2Apresumably can remain in solution longer, due to the stericinhibition of precipitation posed by the bulky diketopiper-azine ring. The pK. for the first pyrrole ionization is 8.50.Spectral fitting (33) using spectra recorded below pH 8.5was used to calculate the individual species spectra; MA isoctahedral, L'Max ` 19; MH-,A has an em.. = 120; whileMH-2A has an em.. = 277. The species having dissociatedpyrrole hydrogens are extremely sensitive to oxidation of thebound cobalt.To investigate the possible hydrolysis of zinc-bound water

in an imidazole ligand field, a study was carried out on theZn(II)-N-methylimidazole system. Although the pyrrolehydrogen has been replaced-by a methyl group, the basicityof the pyridinium nitrogen is not greatly changed. The pK.of imidazole at 25° in 0.16 M KNO3 is 7.125 4- 0.014, whilethepK4of the N-methylimidazole is 7.209 10.008. There-fore, N-methylimidazole should, be representative of imidazolebut will be freespeie trbcomplications associated with thepyrrole hydrogen.

Analysis of the Zn(hi)-N-methylimidazole system providedan unexpected wealth of information (Fig. 2). The stepwisespecies MA through MA4 all maximize within a range of 1.2pH units. Then follows a break of 1.2 pH units before the ap-pearance of MA5anedwih,valueo by a scant 0.24 pH units.Accompanying this unusual pH distribution are "rollercoaster" stability constants (Table 2). The logK values followa repeating pattern of low, high through all six stepwise species,MA4 showing the highest stability. The uniqueness of this

TABLE 1. Summary of the ionization constants

METAL- KaMEON LIGAND (A) SPECIES ION- ~ION__ _ __ _ __ _ _IZATION

Zn (11) IMIDAZOLE MH-iA2 N-H 7.0-7.3

Zn (11) 4,4ABISAIMIDAZOYLMETHANE MH-iA2 N-H 7.90CH2

H' H

Co (11) C-L HISTIDYL-L-HISTIDINE MH-i A N-H 8.50

Zn (11) N-METHYLIMIDAZOLE MH-iA4 M-O 9.12

f-i'

pattern at first caused some concern. However, the furtheraddition of data points did not alter the low, high pattern.As well, the pattern is maintained both in regions of lowerand extremely high experimental accuracy, and is maintainedin MA5 and MA6, which are clearly separated from the pre-ceding species. This finding is not without precedent, sinceseveral zinc systems have been analyzed that do not followthe normal pattern, log K1> log K2> log K3 > log K4 (34-36).

Returning to the question of hydrolysis, it is clearly evidentthat this system extensively, hydrolyzes metal-bound waterat higher pH. The progenitor is MA4, which loses a zinc-bound water proton with a pKa of 9.12. At higher pH a secondbound hydroxide is formed. It should be stressed that thereis no visual, pH drift, or curve-fitting evidence for polymer-ization or for metal hydroxide formation in this system, asfar as it has been studied (pH 11.5). It is of interest to addthat the curve shape for the species MH-jA3 has a pH de-pendence very similar to that described for MH- 1A4.

DISCUSSION

The intrinsic pK. values of water and of neutral imidazolefall within an order of magnitude. Enhancement of the re-spective proton labilities will be expected if the water mole-cule or the imidazole pyridinium nitrogen is bound to the zinc.The question then is one of the relative transmittability ofthe metal ion influence to the two ionizing centers. In thesystems studied, it would appear that the transmittabilityis accentuated in the case of the pyrrole ionization. That is,given a system in which both ionizations can occur, the ion-ization of a pyrrole hydrogen will take precedence over theionization of metal bound water. This is clearly evident sincethe ionization of the bound water can only be studied whenthe pyrrole hydrogen has been replaced by a methyl group.The degree to which the pyrrole ionization is favored varies,

depending on the nature of the complex. The most acidicpyrrole hydrogen is found in the Zn(II)-imidazole system(Table 1). Since the species maximum of MH-jA2 occursfollowing the onset of precipitation, the pKa can be obtainedonly after extrapolation of the curve shape to a maximum.Consequently, the pK. is approximated as lying between7.0 and 7.3. The other two chelates studied in the contextof pyrrole ionization both have progressively less acidic hydro-gens, giving rise to a variation in pKa of about 7 through8.5.

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Activity-Related Ionization in Carbonic Anhydrase 1689

TABLE 2. Stability constants for theZn(HI)-N-methylimidazole 8y8tem*

p q r log IPpqr log K.0 1 1 7.2091 0 1 2.380 K1 = 2.3801 0 2 4.924 K2 = 2.5441 0 3 6.600 Ks= 1.6761 0 4 9.214 K4 = -2. 6141 0 5 10.005 K5=0.7911 0 6 11.047 KG = 1.0421 -1 4 0.1571 --2 4 -10.615

* Log stability constants (log .pqr) of complex species MpHqAr[M = Zn(H), A = N-methylimidazole] in 0.16M KNOs at 250.The log K. values are given as well, where log K. = log 61On-log j#1O(n-1).

Since ionization of the pyrrole hydrogen more or less pre-cludes the ionization of bound water in the same system, itwas necessary to study N-methylimidazole to characterizethe ability of an imidazole chelate system to labilize the hydro-gens on a bound water molecule. The pK. for the ionizationof a water molecule in a zinc complex containing four boundimidazoles was found to be 9.12 (Table 1). This value is suf-ficiently higher than, say, the value of 7.90 obtained with theZn(II)-4,4'-bis-imidazoylmethane to explain the presenceof only the pyrrole ionization in systems which can ionizeeither the N-H or the water. It could be argued that a reduc-tion of the ligand field to three imidazoles may labilize stillfurther the water hydrolysis. To rule this out, the speciesmatrix was solved including MH-1A3. It was found thatMH-1A3 and MH-1A4 have almost identical pK. values,suggesting that the ionization of metal-bound water is notoverly sensitive to changes in the neutral ligand field.Other features of the Zn(II)-N-methylimidazole system

may also prove to be pertinent to a comprehensive under-standing of these ionizations. From the species distribution,considering the high stability of MA4, one is led to believethat most of MA4 is tetrahedral, with a small amount aquatedand octahedral. This small amount of octahedral MA4 wouldthen be the template for the formation of the four higher pHspecies. Possibly, the relatively higher stability of MA2 re-flects the initial change from octahedral to tetrahedral zinc.In any case, the tetrahedral-like environment presented bythe enzyme does coincide with the preferred geometry of zinc,at least in an imidazole ligand field.

In this conjunction it is perhaps worthwhile to point outthat the enzyme, in the protonated state, may have one ormore of the pyrrole hydrogens hydrogen-bonded to adjacentacceptor groups on the protein backbone (13). Subsequentdeprotonation would alleviate this point of attachment, allow-ing the chelate to move towards a more preferred conforma-tion. This would adequately explain the magnitude of thespectral change noted on deprotonation of the cobalt enzyme.

Considering that the hydration of CO2 involves the addi-tion of no less than a hydroxyl ion to the substrate, the forma-tion of a metal-bound hydroxyl ion represents the most com-pelling feature of the ionizing water hypothesis. A mechanismdependent on the ionization of a pyrrole hydrogen must de-velop the nucleophile indirectly. Such an indirect formationcould occur if the imidazolate anion accepted a water proton

H H-- ° C O

0

FIG. 3. A mechanism for the catalysis of C02 hydration by a

zinc-bound anionic imidazole.

locally forming an OH- ion. In Fig. 3 is shown a mechanismthat illustrates how such OH- ion could be used. This mech-anism pertains to the human B enzyme, since Khalifah (3)has shown that imidazole is a competitive inhibitor of theB enzyme, binding directly to the zinc ion (40). This inhibitoris unique, since it does not flood the system with a negativecharge. This would allow the imidazole anion to exist in theabsence of metal bound water.

If the C02 hydrogen-bonded to the water, the pyrrole ion-ization may be perturbed slightly, raising the pK,, causingthe nitrogen to bind a proton and form the nucleophile. Re-cently, Koeftig et al. (41) have presented further evidencesupporting the formation of H2CO3 as product. In this con-

text, the above mechanism is more easily rationalized sincethe metal-bound water would then completely donate theproton, temporarily "inhibiting" the pyrrole ionization.Although such a mechanism is less straightforward, it does

have intriguing possibilities. Unless one anionic pyrrole nitro-gen is specifically stabilized, the net dissociation may extendover the three rings. This "tautomeric set" could facilitatethe use of much of the upper chelate surface for catalysis.To carry the model studies to a logical conclusion, we are

presently synthesizing a triimidazole chelate. This will hope-fully enable us to more fully characterize the tendencies in-herent in non enzymatic systems. Perhaps this work, in con-

junction with further purposeful work on the enzymet, willestablish the true nature of the ionizing group.

This work was supported by a grant from The Medical Re-search Council of Canada.

t Proton magnetic resonance studies on the human B enzymehave shown resonances with chemical shifts characteristic forhistidine C2 protons, which were classed as nontitrating oranomalous (37, 38). Studies in this laboratory on Zn(II)-4,4'-bis-imidazolylmethane solutions indicate that the binding ofZn(II) shifts the free base C2 proton resonances downfield by morethan 0.6 ppm. As well, Pugmire and Grant (39) have shown thatthe change in chemical shift of the C2 proton accompanying thedeprotonation of the free base is only 16% the magnitude of thatseen for the cationic imidazole deprotonation. Therefore, thedownfield resonances in the enzyme spectra must be consideredsuspect, and warrant a close reexamination as to subtle titrationbehavior.

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1. Kernohan, J. C. (1964) Biochim. Biophys. Acta 81, 346-356.

2. Gibbons, B. H. & Edsall, J. T. (1964) J. Biol. Chem. 239,2539-2544.

3. Khalifah, R. G. (1971) J. Biol. Chem. 246, 2561-2573.4. Coleman, J. E. (1967) J. Biol. Chem. 242, 5212-5219.5. Fabry, M. E., Koenig, S. H. & Schillinger, W. E. (1970)

J. Biol. Chem. 245, 4256-4262.6. Lindskog, S., Henderson, L. E., Kannan, K. K., Liljas, A.,

Nyman, P. 0. & Strandberg, B. (1971) in The Enzymes, ed.Boyer, P. D. (Academic Press, New York), Vol. 5, pp.587-665.

7. Lindskog, S. (1970) Struct. Bonding (Berlin), 8, 153-196.8. Lindskog, S. (1963) J. Biol. Chem. 238,945-951.9. Lindskog, S. & Nyman, P. 0. (1964) Biochim. Biophys.

Acta 85, 462-474.10. Coleman, J. E. (1965) Biochemistry 4, 2644-2655.11. Lindskog, S. (1966) Biochim. Biophys. Acta 122, 534-

537.12. Ward, R. L. (1969) Biochemistry 8, 1879-1883.13. Liljas, A., Kannan, K. K., Bergst6n, P.-C., Waara, I.,

Fridborg, K., Strandberg, B., Carlbom, U., JRrup, L.,Lovgren, S. & Petef, M. (1972) Nature New Biol. 235, 131-137.

14. Lindskog, S. & Ehrenberg, A. (1967) J. Mol. Biol. 24, 133-137.

15. Rosenberg, R. C., Root, C. A., Wang, R., Cerdonio, M. &Gray, H. B. (1973) Proc. Nat. Acad. Sci. USA 70, 161-163.

16. Coleman, J. E. & Coleman, R. V. (1972) J. Biol. Chem. 247,4718-4728.

17. Walba, H. & Isensee, R. W. (1956) J. Chem. Soc. 21, 702-704.

18. Chaffee, E., Dasgupta, T. P. & Harris, G. M. (1973) J.Amer. Chem. Soc. 95, 4169-4173.

19. Breslow, E. (1966) in The Biochemistry of Copper, eds.Peisach, J., Aisen, P. & Blumberg, W. E. (Academic Press,New York), pp. 149-157.

20. Prince, R. H. & Woolley, P. R. (1972) Angew. Chem. Int.Ed. Engl. 11, 408-417.

21. Lindskog, S. & Coleman, J. E. (1973) Proc. Nat. Acad. Sci.USA 70, 2505-2508.

22. Koenig, S. H. & Brown, R. D., III (1972) Proc. Nat. Acad.Sci. USA 69, 2422-2425.

23. Fischer, E. & Suzuki, U. (1905) Chem. Ber. 38, 4173-4196.24. Drey, C. N. C. & Fruton, J. S. (1965) Biochemistry 4, 1-5.25. Bates, R. G. (1964) in Determination of pH, Theory and

Practice (John Wiley and Sons, Inc., New York), pp. 123-130.

26. Kruck, T. P. A. & Sarkar, B. (1973) Can. J. Chem. 51, 3549-3554.

27. Sarkar, B. & Kruck, T. P. A. (1973) Can. J. Chem. 51, 3541-3548.

28. Freeman, H. C. (1967) in Advances in Protein Chemistry,eds. Anfinsen, C. B., Jr., Anson, M. L., Edsall, J. T. &Richards, F. M. (Academic Press, New York), Vol. 22,pp. 290-294.

29. Cordes de N. D., M. & Walter, J. L. (1968) Spectrochim.Acta Part A 24, 237-252.

30. Martel, A. E. & Sill6n, L. G. (1964) in Stability Constantsof Metal-Ion Complexes (special publication No. 17, theChemical Society, London).

31. Drey, C. N. C. & Fruton, J. S. (1965) Biochemistry 4, 1258-1263.

32. Ziauddin, Kopple, K. D. & Bush, C. A. (1972) Tetra-hedron Lett. 483-486.

33. Kruck, T. P. A. & Sarkar, B. (1973) Can. J. Chem. 51,3563-3571.

34. Mironov, V. E., Kul'ba, F. Ya., Rutkovskii, Yu. I. &Ignatenko, E. I. (1966) Zh. Neorg. Khim. 11, 955-958.

35. Hershenson, H. M., Brooks, R. H. & Murphy, M. E.(1957) J. Amer. Chem Soc. 79, 2046-2048.

36. Nyman, C. J. (1953) J. Amer. Chem. Soc. 75, 3575-3576.37. King, R. W. & Roberts, G. C. K. (1971) Biochemistry 10,

558-565.38. Cohen, J. S., Yim, C. T., Kandel, M., Gornall, A. G., Kandel,

S. I. & Freedman, M. H. (1972) Biochemistry 11, 327-334.39. Pugmire, R. J. & Grant, D. M. (1968) J. Amer. Chem. Soc.

90, 4232-4238.40. See footnote 3, in ref. 3, p. 2566.41. Koenig, S. H., Brown, R. D., Needham, T. E. & Matwiyoff,

N. A. (1973) Biochem. Biophys. Res. Commun. 53, 624-630.

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