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Stability constant for the zinc-dithiothreitol complex
ANALYTICAL
47, 203-208
BIOCHEMISTRY
Stability
Constant NEAL
Departmed
for
(1972)
the
W. CORNELL of Chemistry,
Pomona Received
Zinc-Dithiothreitol KAREN
AKD
College, August
Complex
E. CRIVARO
Claremont,
California
OlYll
27, 1971
For in vitro studies of biological extracts it is a common practice to use low molecular weight thiols to prevent oxidation of protein sulfhydryl groups. Among such reagents are cysteine, mercaptoethanol, and dithiothreitol. However, it is often not recognized that these low molecular weight thiols also form stable coordination complexes with metal ions (I), and thus possess a second mean of influencing biochemical activities. The purpose of the preesnt paper is to report the stability constants for the zinc complexes of mercaptoethanol and dithiothreitol. The latter was introduced as a sulfhydryl reagent in 1964 (2), and has subsequently become widely used. Our results, however, indicate that dithiothreitol should not be used in systems in which free or accessible Zn+% is involved. The enzyme histidase (L-histidine ammonia-lyase, EC 4.3.1.3) shows t’he behavior characteristic of metalloenzymes, i.e., it is inhibited by a variety of metal binding agents (3,4) and, in the case of rat liver histidase, the inhibition is most effectively reversed by added Znt2 ions (3). On examining these data it seemed that the effectiveness of competitive inhibitors for histidase was correlated with the affinity of the inhibitors for complexed Znt2 ions. The experiments of this report allowed that relationship to be defined and, consequently, provided an enzymic method of determining the stability constants1 for the zinc-mercaptoethanol and zinc-dithiothreitol complexes. MATERIALS
AND
METHODS
Quick-frozen rat livers were obtained from Fel-Freez Biologicals, Inc. (Rogers, Ark.), and were stored at -70” until used. Histidase was prepared by the procedure described previously (3)) and the dialyzed enzyme was divided into l-2 ml fractions, quick-frozen in a dry ice-acetone bath, and stored at -70” with little loss of activity over a two month period. Some material was rendered insoluble by freezing and thawing but could ’ K,t will be used as the stant for reaction of ligand
symbol for stability constant, i.e., the equilibrium and metal ion to give a 1: 1 complex.
@ 1972 by
Inc.
203 Academic
Press,
con-
204
CORNELL
AND
CRIVARO
be removed by centrifugation without affecting the concentration of histidase activity. Potassium pyrophosphate was previously used as the buffer for histidase assays (3), but, since the pyrophosphate complex with Zn+’ has a K,t similar to that for the zinc-histidine complex (log J&t is 6.5 for zinc-pyrophosphate and 6.6 for zinc-histidine) (5)) diethanolamine was substituted in the present experiments. Assays for histidase activity were conducted with a Coleman model 124 spectrophotometer equipped with a thermostated cuvet holder. The standard assay mixture, 390 pmoles of diethanolamine (pH 9.2) and approximately 3 units of histidase activity in H,O so that the total assay volume was 3.0 ml, was preincubated at 37” for 20 min, after which inhibitor (if any) and then substrate (n-histidine, pH 9) were added. The reaction was followed by recording absorbance at 277 nm, the absorption maximum for urocanic acid, the reaction product. Using the molar extinction coefficient of 18800 for the product, 1 unit of histidase activity was defined as the amount catalyzing production of 1 nmole of urocanic acid per minute at 37”. Dithiothreitol, glycine, and dimercaptopropanol were purchased from Calbiochem (Los Angeles), methyliminodiacetic acid from Aldrich (Milwaukee), and other inhibitors from Sigma (St. Louis). Solutions were prepared and glassware was rinsed with distilled water that had been passed through a deionizing column and had a specific conductance of 1 pmho/cm or less. RESULTS
AND
DISCUSSION
Ten compounds which are competitive inhibitors for rat liver histidase were used in this study, and the data obtained are illustrated by those in Fig. 1, a Lineweaver-Burk plot for histidase assays conducted without inhibitor, with cysteine, or with dithiothreitol. As has been reported for the bacterial enzyme (4,6), inhibition by cysteine is competitive with substrate, L-histidine. Dithiothreitol has been reported to activate (6,7), and, at concentrations greater than optimal for activation, to inhibit, bacterial histidase (7). Rat liver histidase, however, when assayed as described in “Materials and Methods,” is only inhibited by dithiothreitol. In addition, it was found that the presence of 20 PLM Zn+2 (added as the acetate salt) in the standard assay completely prevents the inhibitory effect of 1 mM cysteine or 1.7 mM dithiothreitol. Inhibition by metal binding agents and prevention of inhibition by addition of appropriate metal ions is useful in characterizing metalloenzymes (1)) and observation of those properties for rat liver histidase is consistent with our previous suggestion of a role for Zn+2 in substrate binding t’o the enzyme (3). The latter suggestion made it of interest to study the effects of
ZISC-DITHIOTHREITOL
COMPLEX
205
6
4
FIG. 1. Effect of cysteine or dithiothreitol on histidase activity. Assays were conducted as described under “Materials and Methods” either in the absence of inhibitor 0, with 1.0 mM cysteine (A), or with 1.0 mM dithiothreitol (0).
compounds with known metal binding properties, and listed in Table 1 are ten compounds that are competitive inhibitors for rat liver histidase. In all cases, rate data were analyzed both by Lineweaver-Burk and by Hofstee plots (see reference 8 for a discussion of the desirability of using both plots) and the best fitting lines were determined by the method of least squares. No set of dat.a was acceptable unless the product-moment correlation coefficients (9) were > 0.98 for both types of plots. Each KI was calculated from Lineweaver-Burk analysis of at least two sets of data that met this criterion. Eight of the compounds used in our experiments have been characterized as ligands for Zn+2, and the corresponding stability constants are also given in Table 1. While other factors such as stereochemistry might be significant with different systems, with histidase it is apparent that effectiveness of inhibitors is directly related to the inhibitor’s affinity for Zn+2. In fact, as shown by Fig. 2, a plot of K, against log K,t for all inhibitors with K, > 0.1 mM is linear. Points have also been placed on Fig. 2 for mercaptoethano1 and dithiothreitol although, to our
206
CORNELL
K, and h’,~ Values
for
Inhibitor L-Histidine (substrate) Glycine blercaptoethanol D-Histidine Methyliminodiacetic Mercaptoethylamine Cysteine Dithiothreitol Nitrilotriacet,ic acid I)imercaptopropanol Ethylenediaminetetraacetic
AKD
TABLE Compet.itive
CRIVARO
1 Inhibifors K[
acid
acid
0.90 9.10 8.20 6.95 5.45 1.12 1.06 0.38 0.10 0.05 0.01
of Histidase
(InfIr)
log
-
(K,)
u log K,, for mercaptoethanol and dithiothreitol were obtained paper. All other stability const,ants are from reference 5.
h-3,
5.4 5.9Q 6.6 7.6 Y.9 9.9 10.3” 10.5 13.5 16.4 from
Fig.
2 of this
knowledge, stability constants for zinc complexes of these compounds have not been reported. Using the relationship defined by Fig. 2, it is estimated that log Kat is 5.9 for zinc-mercaptoethanol and 10.3 for zincdithiothreitol. These results are in accord with those obtained with two different zinc metalloenzymes. Cysteine and dithiothreito1 were found to be equally effective as inhibitors of phosphomannose isomerase, but to achieve the same degree of inhibition a 7-fold greater concentration of mercaptoethanol was required (10). Likewise, mercaptoethanol was reported to a less effective inhibitor of zinc carboxypeptidase than was either thioglycolate or cysteine, and it was also recognized that effectiveness of inhibitors was directly related to the stability constants of the corresponding zinc complexes (11). Several points should be made concerning the data in Table 1 and Fig. 2. First, although our data were obtained by an enzymic method, a pattern is seen which corresponds to that for data obtained by traditional means. That is, with various bidentate ligands which form complexes consisting of 5-membered rings, replacing oxygen or nitrogen with the highly polarizable sulfur atom leads to enhanced complex stability (1) ; that replacement also leads to greater inhibition of histidase, as can be seen by comparing data for mercaptoethylamine and cysteine with those for glycine and mercaptoethanol. An analogous ligand, dimercaptoethanol, in which both coordination sites are sulfur atoms, is the most effective histidase inhibitor and also forms the most stable zinc complexes. A second point about these data concerns the coordination sites in dithiothreitol The latter, which can be viewed as two linked molecules of mercaptoethanol, is a much more effective enzyme inhibitor. This
ZIR’C-DITHIOTHREITOL
207
CORII’LES
9
7
Log
Stability
Constant
FIG. 2. Relationship between K, and K,t. Assays and calculations were performed as described in the text. Inhibitors included in this graph are: glycine (A). mercaptoethanol (B), n-histidine (C), methyliminiodiacetic acid (D), mercaptoethylamine (E), cysteine (F), dithiothreitol (G), and nitrilotriacetic acid (H).
suggest’sthat dithiothreitol cyclizes as it does on oxidation (2) and coordinates with zinc through its two sulfurs, forming a 7-membered OH
,s"Ln-S,
-S-CH,-l!H-CH-CH,-SH bH
t
Zn-'-
CH, \L
CHz
f
"+
ring.2 Since decreased stability usually accompanies an increase in ring size of complexes (13)) a smaller log K,, would be expected for Zndithiothreitol relative to that for the dimercaptopropanol complex; that expectation is met by our results. Finally, although the histidase assay conditions (pH 9.2, protein solution, slightly variable ionic strengths) arc atypical for measurements of stability constants, the log li,t values for 6 compounds plotted in Fig. 2 “On the basis of our titration data, dithiothreitol is written as partially ionized. Titrations were performed in H1O, under N,, with KOH, and the ionization constants were computed as dcscribcd by Blbert and Srrgexnt (12) for &basic acids with overlapping pK, values. Results for eight titrations were: pK,, = 9.1 k 0.02 and assay, the two -SH groups pK,, = 9.8 f 0.1. Thus, at pH 9.2 used in the histidase of dithiothreitol would be 56% and 20% ionized.
208
CORNELL
AiXD
CRIVARO
were determined under defined chemical conditions. Consequently the estimate of log list = 10.3 for the zinc-dithiothreitol complex would appear to be free of any large error due to the assay conditions, and use of alternative thiols is indicated for biological systems requiring Zn+2 ions. Complexing of t’he biochemically more common ions, Mn+? and Mg’“, by dithiothreitol remains to be studied, but, by comparisons with numerous ligands (1,5), it is expected that log I&t would be on the order of 4 for the Mn-dithiothreitol complex and <4 for the Mg-dithiothreitol complex. SCMMARY
Rat liver histidase is competitively inhibited by a variety of compounds that are good complexing agents for Zn+“. Dithiothreitol is a strong competitive inhibitor, but. 20 PJI Zn+2 completely reverses the inhibitory action of 1.7 mM dithiothreitol. Effectiveness of inhibitors is directly related to the stability constants (I&) for the corresponding zinc complexes. A plot of KI against log K,t is linear, and was used to estimate log KSt for the zinc-dithiothreitol complex. A value of 10.3 was obtained, indicating that dithiothreitol should not be used in biological systems in which free or accessible Zn+2 is involved. Mercaptoethanol was characterized in the same manner, and log KS, was found to be 5.9 for its complex with zinc. ACKNOWLEDGMENT This Health.
work
was
supported
by
grant
HD
03620
from
the
National
Institutes
of
REFERENCES 1. VALLEE, B. I,., AND WACKER. W. C., in “The Proteins” (H. Neurath, ed.), Vol. V,, Chap. 6. Academic Press, New York, 1970. 2. CLELAND, W. W., BiochewLstry 3, 480 (1964). 3. CORNELL, K. W., AND LIEN, L. L., Physiol. Chem. Phys. 2, 523 (1970). 4. PETERKOFSKY, A., AND MEHLER, L. N., Biochim. Biophgs. Acfa 73, 159 (1963). 5. SILLEpi. 1,. G., ASD MARTELL, A. E., in “Stability Constants of Metal-Ion Complexes.” Chemical Society (London), Spec. Publ. No. 17 (1964). 6. RECHLER, M. M., J. Biol. Chem. 244, 551 (1969). 7. SMITH, T. A., CORDELLE, F. H., AND ARELES, R. H., Arch. Biochem. Biophys. 120, 724 (1967). S. DOIVD, .J. E.. AND Rrnan, D. S., J. Biol. Chrm. 240, 863 (1965). 9. SOKAL. R. R., .~NU ROHLF, F. J., ia “Biometry.” Freeman, San Francisco, 1969. 10. GRACY, R. W., AND NOLTMANN, E. A., J. Biol. Chem. 243, 4109 (1969). 11. COOMBS, T. L., FELBER, J. P., AND VALLEE, B. L., Biochemistry 1, 899 (1962). 12. ALBERT, A., AND SERGEANT, E. P., in “Ionization Constants of Acids and Bases.” Wiley, New York, 1962. 13. ROSWI-TI, F. J. C., iu “Modern Coordination Chemistry” (J. I,cwis and R. G. Wilkins, eds.), Chap. 1. Interscience, New York, 1960.
47, 203-208
BIOCHEMISTRY
Stability
Constant NEAL
Departmed
for
(1972)
the
W. CORNELL of Chemistry,
Pomona Received
Zinc-Dithiothreitol KAREN
AKD
College, August
Complex
E. CRIVARO
Claremont,
California
OlYll
27, 1971
For in vitro studies of biological extracts it is a common practice to use low molecular weight thiols to prevent oxidation of protein sulfhydryl groups. Among such reagents are cysteine, mercaptoethanol, and dithiothreitol. However, it is often not recognized that these low molecular weight thiols also form stable coordination complexes with metal ions (I), and thus possess a second mean of influencing biochemical activities. The purpose of the preesnt paper is to report the stability constants for the zinc complexes of mercaptoethanol and dithiothreitol. The latter was introduced as a sulfhydryl reagent in 1964 (2), and has subsequently become widely used. Our results, however, indicate that dithiothreitol should not be used in systems in which free or accessible Zn+% is involved. The enzyme histidase (L-histidine ammonia-lyase, EC 4.3.1.3) shows t’he behavior characteristic of metalloenzymes, i.e., it is inhibited by a variety of metal binding agents (3,4) and, in the case of rat liver histidase, the inhibition is most effectively reversed by added Znt2 ions (3). On examining these data it seemed that the effectiveness of competitive inhibitors for histidase was correlated with the affinity of the inhibitors for complexed Znt2 ions. The experiments of this report allowed that relationship to be defined and, consequently, provided an enzymic method of determining the stability constants1 for the zinc-mercaptoethanol and zinc-dithiothreitol complexes. MATERIALS
AND
METHODS
Quick-frozen rat livers were obtained from Fel-Freez Biologicals, Inc. (Rogers, Ark.), and were stored at -70” until used. Histidase was prepared by the procedure described previously (3)) and the dialyzed enzyme was divided into l-2 ml fractions, quick-frozen in a dry ice-acetone bath, and stored at -70” with little loss of activity over a two month period. Some material was rendered insoluble by freezing and thawing but could ’ K,t will be used as the stant for reaction of ligand
symbol for stability constant, i.e., the equilibrium and metal ion to give a 1: 1 complex.
@ 1972 by
Inc.
203 Academic
Press,
con-
204
CORNELL
AND
CRIVARO
be removed by centrifugation without affecting the concentration of histidase activity. Potassium pyrophosphate was previously used as the buffer for histidase assays (3), but, since the pyrophosphate complex with Zn+’ has a K,t similar to that for the zinc-histidine complex (log J&t is 6.5 for zinc-pyrophosphate and 6.6 for zinc-histidine) (5)) diethanolamine was substituted in the present experiments. Assays for histidase activity were conducted with a Coleman model 124 spectrophotometer equipped with a thermostated cuvet holder. The standard assay mixture, 390 pmoles of diethanolamine (pH 9.2) and approximately 3 units of histidase activity in H,O so that the total assay volume was 3.0 ml, was preincubated at 37” for 20 min, after which inhibitor (if any) and then substrate (n-histidine, pH 9) were added. The reaction was followed by recording absorbance at 277 nm, the absorption maximum for urocanic acid, the reaction product. Using the molar extinction coefficient of 18800 for the product, 1 unit of histidase activity was defined as the amount catalyzing production of 1 nmole of urocanic acid per minute at 37”. Dithiothreitol, glycine, and dimercaptopropanol were purchased from Calbiochem (Los Angeles), methyliminodiacetic acid from Aldrich (Milwaukee), and other inhibitors from Sigma (St. Louis). Solutions were prepared and glassware was rinsed with distilled water that had been passed through a deionizing column and had a specific conductance of 1 pmho/cm or less. RESULTS
AND
DISCUSSION
Ten compounds which are competitive inhibitors for rat liver histidase were used in this study, and the data obtained are illustrated by those in Fig. 1, a Lineweaver-Burk plot for histidase assays conducted without inhibitor, with cysteine, or with dithiothreitol. As has been reported for the bacterial enzyme (4,6), inhibition by cysteine is competitive with substrate, L-histidine. Dithiothreitol has been reported to activate (6,7), and, at concentrations greater than optimal for activation, to inhibit, bacterial histidase (7). Rat liver histidase, however, when assayed as described in “Materials and Methods,” is only inhibited by dithiothreitol. In addition, it was found that the presence of 20 PLM Zn+2 (added as the acetate salt) in the standard assay completely prevents the inhibitory effect of 1 mM cysteine or 1.7 mM dithiothreitol. Inhibition by metal binding agents and prevention of inhibition by addition of appropriate metal ions is useful in characterizing metalloenzymes (1)) and observation of those properties for rat liver histidase is consistent with our previous suggestion of a role for Zn+2 in substrate binding t’o the enzyme (3). The latter suggestion made it of interest to study the effects of
ZISC-DITHIOTHREITOL
COMPLEX
205
6
4
FIG. 1. Effect of cysteine or dithiothreitol on histidase activity. Assays were conducted as described under “Materials and Methods” either in the absence of inhibitor 0, with 1.0 mM cysteine (A), or with 1.0 mM dithiothreitol (0).
compounds with known metal binding properties, and listed in Table 1 are ten compounds that are competitive inhibitors for rat liver histidase. In all cases, rate data were analyzed both by Lineweaver-Burk and by Hofstee plots (see reference 8 for a discussion of the desirability of using both plots) and the best fitting lines were determined by the method of least squares. No set of dat.a was acceptable unless the product-moment correlation coefficients (9) were > 0.98 for both types of plots. Each KI was calculated from Lineweaver-Burk analysis of at least two sets of data that met this criterion. Eight of the compounds used in our experiments have been characterized as ligands for Zn+2, and the corresponding stability constants are also given in Table 1. While other factors such as stereochemistry might be significant with different systems, with histidase it is apparent that effectiveness of inhibitors is directly related to the inhibitor’s affinity for Zn+2. In fact, as shown by Fig. 2, a plot of K, against log K,t for all inhibitors with K, > 0.1 mM is linear. Points have also been placed on Fig. 2 for mercaptoethano1 and dithiothreitol although, to our
206
CORNELL
K, and h’,~ Values
for
Inhibitor L-Histidine (substrate) Glycine blercaptoethanol D-Histidine Methyliminodiacetic Mercaptoethylamine Cysteine Dithiothreitol Nitrilotriacet,ic acid I)imercaptopropanol Ethylenediaminetetraacetic
AKD
TABLE Compet.itive
CRIVARO
1 Inhibifors K[
acid
acid
0.90 9.10 8.20 6.95 5.45 1.12 1.06 0.38 0.10 0.05 0.01
of Histidase
(InfIr)
log
-
(K,)
u log K,, for mercaptoethanol and dithiothreitol were obtained paper. All other stability const,ants are from reference 5.
h-3,
5.4 5.9Q 6.6 7.6 Y.9 9.9 10.3” 10.5 13.5 16.4 from
Fig.
2 of this
knowledge, stability constants for zinc complexes of these compounds have not been reported. Using the relationship defined by Fig. 2, it is estimated that log Kat is 5.9 for zinc-mercaptoethanol and 10.3 for zincdithiothreitol. These results are in accord with those obtained with two different zinc metalloenzymes. Cysteine and dithiothreito1 were found to be equally effective as inhibitors of phosphomannose isomerase, but to achieve the same degree of inhibition a 7-fold greater concentration of mercaptoethanol was required (10). Likewise, mercaptoethanol was reported to a less effective inhibitor of zinc carboxypeptidase than was either thioglycolate or cysteine, and it was also recognized that effectiveness of inhibitors was directly related to the stability constants of the corresponding zinc complexes (11). Several points should be made concerning the data in Table 1 and Fig. 2. First, although our data were obtained by an enzymic method, a pattern is seen which corresponds to that for data obtained by traditional means. That is, with various bidentate ligands which form complexes consisting of 5-membered rings, replacing oxygen or nitrogen with the highly polarizable sulfur atom leads to enhanced complex stability (1) ; that replacement also leads to greater inhibition of histidase, as can be seen by comparing data for mercaptoethylamine and cysteine with those for glycine and mercaptoethanol. An analogous ligand, dimercaptoethanol, in which both coordination sites are sulfur atoms, is the most effective histidase inhibitor and also forms the most stable zinc complexes. A second point about these data concerns the coordination sites in dithiothreitol The latter, which can be viewed as two linked molecules of mercaptoethanol, is a much more effective enzyme inhibitor. This
ZIR’C-DITHIOTHREITOL
207
CORII’LES
9
7
Log
Stability
Constant
FIG. 2. Relationship between K, and K,t. Assays and calculations were performed as described in the text. Inhibitors included in this graph are: glycine (A). mercaptoethanol (B), n-histidine (C), methyliminiodiacetic acid (D), mercaptoethylamine (E), cysteine (F), dithiothreitol (G), and nitrilotriacetic acid (H).
suggest’sthat dithiothreitol cyclizes as it does on oxidation (2) and coordinates with zinc through its two sulfurs, forming a 7-membered OH
,s"Ln-S,
-S-CH,-l!H-CH-CH,-SH bH
t
Zn-'-
CH, \L
CHz
f
"+
ring.2 Since decreased stability usually accompanies an increase in ring size of complexes (13)) a smaller log K,, would be expected for Zndithiothreitol relative to that for the dimercaptopropanol complex; that expectation is met by our results. Finally, although the histidase assay conditions (pH 9.2, protein solution, slightly variable ionic strengths) arc atypical for measurements of stability constants, the log li,t values for 6 compounds plotted in Fig. 2 “On the basis of our titration data, dithiothreitol is written as partially ionized. Titrations were performed in H1O, under N,, with KOH, and the ionization constants were computed as dcscribcd by Blbert and Srrgexnt (12) for &basic acids with overlapping pK, values. Results for eight titrations were: pK,, = 9.1 k 0.02 and assay, the two -SH groups pK,, = 9.8 f 0.1. Thus, at pH 9.2 used in the histidase of dithiothreitol would be 56% and 20% ionized.
208
CORNELL
AiXD
CRIVARO
were determined under defined chemical conditions. Consequently the estimate of log list = 10.3 for the zinc-dithiothreitol complex would appear to be free of any large error due to the assay conditions, and use of alternative thiols is indicated for biological systems requiring Zn+2 ions. Complexing of t’he biochemically more common ions, Mn+? and Mg’“, by dithiothreitol remains to be studied, but, by comparisons with numerous ligands (1,5), it is expected that log I&t would be on the order of 4 for the Mn-dithiothreitol complex and <4 for the Mg-dithiothreitol complex. SCMMARY
Rat liver histidase is competitively inhibited by a variety of compounds that are good complexing agents for Zn+“. Dithiothreitol is a strong competitive inhibitor, but. 20 PJI Zn+2 completely reverses the inhibitory action of 1.7 mM dithiothreitol. Effectiveness of inhibitors is directly related to the stability constants (I&) for the corresponding zinc complexes. A plot of KI against log K,t is linear, and was used to estimate log KSt for the zinc-dithiothreitol complex. A value of 10.3 was obtained, indicating that dithiothreitol should not be used in biological systems in which free or accessible Zn+2 is involved. Mercaptoethanol was characterized in the same manner, and log KS, was found to be 5.9 for its complex with zinc. ACKNOWLEDGMENT This Health.
work
was
supported
by
grant
HD
03620
from
the
National
Institutes
of
REFERENCES 1. VALLEE, B. I,., AND WACKER. W. C., in “The Proteins” (H. Neurath, ed.), Vol. V,, Chap. 6. Academic Press, New York, 1970. 2. CLELAND, W. W., BiochewLstry 3, 480 (1964). 3. CORNELL, K. W., AND LIEN, L. L., Physiol. Chem. Phys. 2, 523 (1970). 4. PETERKOFSKY, A., AND MEHLER, L. N., Biochim. Biophgs. Acfa 73, 159 (1963). 5. SILLEpi. 1,. G., ASD MARTELL, A. E., in “Stability Constants of Metal-Ion Complexes.” Chemical Society (London), Spec. Publ. No. 17 (1964). 6. RECHLER, M. M., J. Biol. Chem. 244, 551 (1969). 7. SMITH, T. A., CORDELLE, F. H., AND ARELES, R. H., Arch. Biochem. Biophys. 120, 724 (1967). S. DOIVD, .J. E.. AND Rrnan, D. S., J. Biol. Chrm. 240, 863 (1965). 9. SOKAL. R. R., .~NU ROHLF, F. J., ia “Biometry.” Freeman, San Francisco, 1969. 10. GRACY, R. W., AND NOLTMANN, E. A., J. Biol. Chem. 243, 4109 (1969). 11. COOMBS, T. L., FELBER, J. P., AND VALLEE, B. L., Biochemistry 1, 899 (1962). 12. ALBERT, A., AND SERGEANT, E. P., in “Ionization Constants of Acids and Bases.” Wiley, New York, 1962. 13. ROSWI-TI, F. J. C., iu “Modern Coordination Chemistry” (J. I,cwis and R. G. Wilkins, eds.), Chap. 1. Interscience, New York, 1960.