Adventitious inhibition of yeast alcohol dehydrogenase

ARCHIVES

OF

BIOCHEMISTRY

Adventitious FREDERIC

From the Biophysics

AND

BIOPHYSICS

inhibition

L. HOCH,

166-172 (1960)

91,

of Yeast Alcohol

Dehydrogenase’

ROBERT G. MARTIN, WARREN AND BERT L. VALLEE

E. C. WACKER

Research Laboratory of the Department of Medicine, Harvard the Peter Bent Brigham Hospital, Boston, Massachusetts Received

August

Medical

School, and

12, 1960

The inhibition of yeast alcohol dehydrogenase by N’-methylnicotinamide has been shown to be due to contaminating silver ions present in commercial preparations of this reagent. Contamination by metal ions, which was assessed by quantitative analysis, also accounts for the inhibition of yeast alcohol dehydrogenase by semicarbaaide. Purification of the reagents by removal of the metals abolished the inhibition. The existence of enzyme concentration-dependent inhibition serves as a kinetic indicator of such an adventitious inhibition. INTRODUCTION

Yeast alcohol dehydrogenase is inhibited by several classes of reagents: metal ions (l-3), sulfhydryl (3-7), chelating (8) and denaturing reagents (7), and chemical analogs of DPN2 (9-11) or substrates (5). Mechanisms of enzymic catalysis have been inferred from the inhibition of enzymic activity by compounds related in structure to DPN (12-14). It has been recognized that water may be a source of metal-ion contamination which brings about inhibition of yeast alcohol dehydrogenase. The purification of the water or the addition of low concentrations of metal-binding agents resulted in an increase in activity (15, 16). In the course of studies on the inhibition of yeast alcohol dehydrogenase it was observed that traces of metal ions may also be introduced as contaminants of organic reagents studied as inhibitors. In fact, the metal ions produced an adventi1 Supported by the Howard Hughes Medical Institute, and a grant-in-aid from the National Institutes of Health, Department of Health, Education and Welfare, No. H3117(C), and the Nutrition Foundation. 2 The abbreviations used are: DPN, diphosphopyridine nucleotide; DPNH, reduced diphosphopyridine nucleotide; AMP, adenosine monophosphate; ATP, adenosine diphosphate. 166

tious inhibition initially attributed to these organic reagents, as was apparent from the diminution of the inhibition as the metals were removed. Certain anomalies of the resulting kinetics of such adventitious inhibitions are characteristic and may be used to detect the existence of inhibiting contaminants. The inhibit’ions of yeast alcohol dehydrogenase resulting from t’he contamination of N’-methylnicotinamide with silver, and of semicarbazide with copper have been studied in detail. A preliminary account has been given (17). METHODS

AND

MATERIALS

Twice-crystallized yeast alcohol dehydrogenase (18) was obtained from C. F. Boehringer and Sons, Mannheim, W. Germany, and had a turnover number of approximately 30?000 moles DPN/min./ mole enzyme at pH 8.8, 23”, in a reaction mixture containing 1.7 X 1O-3 M DPN and 0.33 M ethanol. The enzyme was dialyzed against 0.1 M phosphate buffer, pH 7.5, 4”, before use. N’-Methylnicotinamide chloride (W. A. Taylor and Company), nicotinamide (Nutritional Biochemicals, Inc.), adenine and adenosine (General Chemicals, Inc.), AMP and ATP (Pabst Laboratories), and semicarbazide base (Fluka, Inc., Switzerland) were neutralized before use. In initial experiments, these reagents were employed without further purification. To remove metals, solutions of the commercial A;‘-methylnicotinamide, in small

ADVENTITIOUS volumes of hot methanol, were filtered through Whatman No.. 50 paper and recrystallized by addition of cold ethyl ether at 4”. The crystals were washed with cold ether. Commercial N’-methylnicotinamide was also treated by adsorption with Norit A charcoal (19). N’-Methylnicotinamide concentrations were determined by absorbancy at 265 rnp using an absorbancy index of 4.5 X lo3 moles/sq. cm. Semicarbaxide hydrochloride was crystallized from the base in metal-free HCl and water. Urea (Merck, analytical grade) was recrystallized after treatment with Amberlite MB-l resin (20). Analytical-grade metal salts were used, and all glassware and water were rendered metalfree (8). Enzymic activity, initiated by the addition of 1-12 pg. enzyme to the remainder of the 3.0.ml. reaction mixture, was measured as described (8). The reaction mixture was buffered at, pH 7.5 with 0.048 M phosphate (analytical grade), at pH 8.8 with 0.048 M pyrophosphate (analytical grade), or at pH 7.5 with 0.033 M tris(hydroxymethyl)aminomethane (primary acidimetric standard grade, Sigma Chemical Co.). Spectrographic analysis of these buffers demonstrated the absence of significant, amounts of metals. Activity, v, is the change in absorbance at 340 mp/min./mg. enzyme; partial activity, v;/v, , is the ratio of inhibited to csontrol activity. Instantaneous (21) inhibitions were examined by adding the enzyme to the reaction mixture containing the various compounds examined. Time-dependent (22) inactivations were examined by preincubating a mixture of bujTer, enzyme, and the suspected inhibitory agent, and removing 0.2-ml. aliquots at measured time intervals for introduction into a 3.ml. reaction mixture containing 1.7 x 10-a M DPN, 0.33 M ethanol, and 0.048 M pyrophosphate buffer, pH 8.8, 23”, or 0.033 M tris(hydroxymethyl)aminomethane buffer, pH 7.5, 23”. Argento-amperometric titrations (20) were performed on yeast alcohol dehydrogenase for

167

INHIBITION 1.0

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FIG. 1. The effect of yeast alcohol dehydrogenase concentration upon the degree of instantaneous inhibition by N’-methylnicotinamide (open symbols) and by silver nitrate (closed symbols). Partial specific activity, vi/v, , is plotted versus the negative logarithm of the concentration of the inhibitor. Activity measurements: DPN, 1.67 X 1OF M; ethanol, 0.33 IV; for N’-methylnicotinamide, 0.048M phosphate buffer, pH 7.5, 23” is used; enzyme is added last to start the reaction, 3.3 X KY9 M (0); 1.3 X 10-8 M (A), and 2.6 X lOwa M (0); for silver nitrate, 0.033 M tris(hydroxymethyl)aminomethane buffer, pH 7.5, 23” is used; enzyme concentrations are 4.5 X 1OF M (a), and 1.3 X 10-8M (A).

bition produced by N’-methylnicotinamide, the most potent inhibitor, was studied in detail, and preliminary observations were made on AMP and ATP. The degree of inhibition observed depends not only upon the concentration of N’-methylnicotinamide, but also upon the concentration of the enzyme (Fig. l), although the former exceeds the latter by a factor of about 105. Inhibition increases as enzyme concenthe estimation of the sulfhydryl groups of the tration decreases. The slopes of the inhibiprotein. The metal content of reagents was meastion curves are steep, and inhibition becomes ured spectrographically (23, 24); silver and copper complete within a tenfold range of N’-methwere determined chemically (25). ylnicotinamide concentration. Though N’methyhCcotinamide is structurally analoRESULTS gous to DPN, this inhibition is noncompetiINHIBITION BY N'-METHYLNICOTINAMIDE tive bot,h with DPK and ethanol; it is not reversible by dilution of the enzyme-inInitial studies confirmed (12-14) that hibitor complex, or by the criteria of Ackeryeast alcohol dehydrogenase is inhibited man and Potter (26). instantaneously by a number of compounds, These phenomena are reminiscent of the structurally analogous to moieties of DPN: N’-methylnicotinamide, AMP, ATP, ade- inhibiting action of metal ions, such as silver, which have a high affinity for sulfhydryl nine, adenosine, and nicotinamide, which enzymes, e.g., yeast alcohol dehydrogenase decrease in efficacy in that order. The inhi-

168

HOCH,

TABLE

MSRTIN,

WACKER

( Aga / Ba

Moles of metal

OF

1 Ca / Cd’ / Fe / Mg

I i I I I I

(X 109 15 f).oo7 Mole of N’-Methylnicotinamide

l.?.13

l.jo.20

all others are spectro0 Chemical analyses; graphic analyses. Not detected were: Al, Be, Ca, Co, Cr, Hg, Li, MO, Mn, Ni, Pb, Sr, V, Zn.

(7). The instantaneous inhibition of the enzyme by low concentrations of silver nitrate, shown in Fig. 1 for comparison, documents this analogy : The irreversible inhibition, noncompetitive with DPN or ethanol (7), is dependent on enzyme concentration, with inhibition curves of slope similar to those with N’-methylnicotinamide. Accordingly, the commercial preparation of N’-methylnicotinamide used in these experiments was analyzed for metals spectrographically and chemically (Table I). Silver is the most significant contaminant. A solution of 1OF M N’-methylnicotinamide is 1.5 X 10e7 M with respect to silver, a concentration of silver which is inhibitory under these conditions (Fig. 1). The silver content of this preparation of commercial N’-methylincotinamide, 1.5 X TABLE TITRATABLE SULFHYDRYL DEHYDROGENASE

VALLEE

lop4 M per mole, could be decreased to 3.0 X 10B5 mole/mole by sixfold recrystallization, or to 5.0 X lo+ mole/mole by adsorption with Norit A charcoal (19). In spite of strenuous efforts, it proved impossible to remove the last vestige of the contaminating silver. The low, residual silver content of the purified material still accounts for the observed, irreversible, enzyme concentrationdependent inhibition, which is now observed at correspondingly higher concentrations of N’-methylnicotinamide. If the observed inhibitsions can actually be attributed to the contaminating silver ions, it would be expected that the free sulfhydryl groups of yeast alcohol dehydrogenase are decreased in proportion to the inhibition. This expectation is borne out, as seen in Table II which compares the per cent decrease of the argentometrically titratable -SH groups with the corresponding decrease of the activity of the enzyme in the presence of commercial and recrystallized N’-methylnicotinamide. The silver content of each of these preparations is also correlated with the concomitant decrease in titratable -SH groups and activity, and even the inhibition by bhe purified material appears to be due to the contaminant. The commercial preparations of AMP and ATP used in the inhibition studies were also analyzed for metal content; the significant findings in moles of metal per lo5 moles of reagent were, AMP: silver 0.21; copper 1.1; iron 1.4; ATP: silver 2.3; copper 3.9; iron

I

SPECTROGRAPHIC AND CHEMICAL A~VALYSES OF A COMIJIERCIAL PREPARATION N’-METHYLNICOTINAUIDE

AND

II

GROUPS AND ENZYMIC ACTIVITY OF YEAST IN THE PRESENCE OF N’-METHYLNICOTINAMIDE

ALCOHOL

N’-Methylnicotinamide Preparation

Commercial

6X crystallized

Concentration

M x 108 0.27 0.50 1.0 0.45 0.88 2.3 4.5

Silver content

-SH content --SH + A”-methylnicotinamide --SH control Sdi/SE,

M x 108

Activity

SilVc

4.0 7.5 15

0.73 0.36 0.12

0.70 0.30 0.05

1.4 2.6 6.9 14

0.95 0.73 0.45 0.16

1.00 0.85 0.31 0.11

ADVENTITIOUS

2.3; chromium 0.69. The inhibition observed with AMP, the next most potent inhibitor of the enzyme, was examined for some of the features observed with IV-methylnicotinamide. Figure 2 demonstrates that the inhibition by the commercial reagent is similarly dependent upon enzyme concentration. This inhibition (and that by ATP) was noncompetitive with DPN and ethanol. However, the AMP inhibition is reversible (26), suggesting Dhat, while these effects may be adventitious, they are not due to an ion act’ing like silver. IIVHIBITIOK

169

INHIBITION

hibition is due to the presence of metal ions which might combine with sulfhydryl groups. Spectrographic and chemical analyses

BY SEMICARBAZIDE

The inhibition of yeast alcohol dehydrogenase by semicarbazide base is time-dependent and increases as a function of the duration of their contact. The first-order rate of this inhibition depends on the concentration of enzyme (Fig. 3), a kinetic anomaly analogous to those observed with N’-methylnicotinamide and AMP. Instantaneous inhibition is not observed. Even though t,he effective molar concentration of the commercial semicarbazide exceeds that of the enzyme by a factor of 106, the rate of inactivat)ion increases as the enzyme concentration decreases. While the initial rates are all first-order, after 50 and 40 min. of incubation, respectively, the rate of inactivation increases when the enzyme is 4 X lo+ M and 2 X 1O-s M; under these conditions this rate increase was observed uniformly. The addition of 10-3 1M ethylenediamine tetracetate or reduced glutathione to semicarbazide prior to the addition of yeast alcohol dehydrogenase prevents inactivation. Dilution, or addition of these reagents after enzyme and semicarbazide base have been in contact, arrests the loss of activity but will not restore it. It might be expected that this time-dependent and enzyme concentration-dependent inhibition is accompanied by a proportionate decrease in the number of titratable sulfhydryl groups of the enzyme, by analogy with Table II. In Fig. 4, the first-order rate of the acbivity loss and the rate of decrease in the per cent of titratable sulfhydryl groups are virtually identical; this is therefore consistent with the hypothesis t,hat the in-

- log (AMP1

FIG. genase taneous ments: enzyme 1OW M) (0).

M

2. The effect of yeast alcohol dehydroconcentration upon the degree of instaninhibition by AMP. Activity measureas in Fig. 1, with N’-methylnicotinamide; is added last to start the reaction, (3.3 X (0), 1.3 X 10-a M (A), and 2.6 X 1O-8 44

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Dumflon of fncubafion

0

6012

-

(Minutes)

FIG. 3. The effect of yeast alcohol dehydrogenase concentration upon the rate of the timedependent inhibition by a commercial preparation of semicarbazide base. The logarithm of the enzymic activity, ZJ, is plotted versus the duration of incubation. Preincubation: 0.08 M semicarbazide, in 0.1 M phosphate buffer, pH 7.5, 23”; control enzyme (0) is 6 X lo-* M, and the other enzyme (YADH) concentrations are indicated. Activity measurements: 0.2 ml. of the incubation mixture is transferred to a reaction mixture as in Fig. 1, 0.048 A4 phosphate buffer, pH 7.5, 23”, to initiate the enzymic reaction.

170

HOCH,

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I

I

MARTIN,

WACKER

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Duration of Incubation (Minutes) FIG. 4. The effect of semicarbazide base upon the activity and titratable sulfhydryl content of yeast alcohol dehydrogenase. The logarithms of the partial activity (0) and the partial sulfhydryl content (X) are plotted versus the duration of incubation in hours. Preincubation: 0.2 M commercial semicarbazide base, in 0.1 M tris(hydroxymethyl)aminomethane buffer, pH 7.5, 23”; 2 X lo-? M enzyme. Aliquots are transferred for measurements of both activity and sulfhydryl groups (20) in 0.033 M tris(hydroxymethyl)aminomethane buffer as in Fig. 3, since the -SH titrations cannot be performed in phosphate buffers.

i,“ri,tbn

of Incubation (Minutes)

5. The effects of recrystallized semicarbazide hydrochloride, and ferrous and cupric salts upon the rate of the time-dependent inhibition of yeast alcohol dehydrogenase. Preincubation: the control enzyme (2 X lo-7 M) (A, 0); the enzyme plus 0.4 M semicarbazide hydrochloride (B, X); plus 3 X 10-’ M ferrous ammonium sulfate and 3 X low7 M cupric chloride (C, A); and enzyme plus 0.4 M semicarbazide hydrochloride and 3 X 1OV M cupric chloride (D, 0); are all incubated, and activity is measured, as in Fig. 3. FIG.

AND

VALLEE

were therefore performed on the commercial semicarbazide base used. It contains only 8 X 10-T mole iron, and 7.5 X lo-’ mole copper per mole. Both iron and copper are present in amounts which would result in concentrations of about 3 X lo--’ M in a solution 0.4 M with respect to semicarbazide. Such low concentrations of ferrous ammonium sulfate and cupric chloride do not, however, result in a t’ime-dependent inhibition (Fig. 5, line C). On the other hand, purification of the commercial semicarbazide base, by recrystallization as the hydrochloride, lowers its metal content below t’he limit of detection by the spectrographic method; a 0.4 M solution of this recrystallized prepation does not inhibit after 150 min. of incubation (Fig. 5, line B) compared to a 94 % inactivation by the unpurified base in the same time. Incubation of the enzyme with 0.4 M semicarbazide hydrochloride and 3 X lo--’ M ferrous iron does not evoke a greater rate of inactivation than does semicarbazide alone. However, when both 3 X lo-’ II/I cupric chloride and 0.4 M recrystallized semicarbazide hydrochloride are incubated together with the enzyme, it is inactivated rapidly (Fig. 5, line D). Additions of reduced glutathione or ethylenediamine tetracetate prevent or arrest these inhibitions. These low concentrations of cupric ions thus become inhibitory only in the presence of semicarbazide hydrochloride; neither the metal ion nor the organic reagent separately inhibit to the degree observed when they are combined. Enhancement of metal-ion inhibition is not confined to semicarbazide. The preincubation of the enzyme with 0.4 M urea for 180 min. does not inhibit its activity significantly; however, the combination of 3 X 10-’ ferrous and cupric salts and 0.4 M urea inhibits 50%. As wit’h semicarbazide, the activity decreases at a first-order rate. DISCUSSION

Low concentrations of ferrous, cupric, silver, mercuric, or zinc ions inhibit crystalline yeast alcohol dehydrogenase (l-3, 7, 15), indicating their high affinity for this enzyme. The source of the significant amounts of metals in the analytical-grade

ADVENTITIOUS

reagents used here obviously may vary. Nor can the degree of contamination of any given reagent be predicted or generalized; metal ions are ubiquitously distributed in natural biological products, they are frequently used in the synthesis of organic chemicals, and they have high affinities for many compounds used as inhibitors of enzymic activities. Insufficiently purified water is now well recognized to be an important source of traces of metal ions inhibitory to yeast alcohol dehyclrogenase, as was demonstrated by the reversal of a previously unsuspected inhibition upon the addition of low concentrations of chelating agents (15, 16). In contrast, the presence of contaminants in N’-methylnicotinamide was initially inferred from demonstrations of anomalous inhibitions. The kinetics of such inhibitions, which are influenced by the concentration of the enzyme in simple enzyme-inhibitor interactions, have been detailed by Straus and Goldstein (27, 28) and apply to both instantane0u.s and time-dependent inhibitions. Significant concentrations of metals were subsequently detected by analytical means. The anomalous inhibition by w-methylnicotinamide appears to be accounted for by its content of silver employed in its synthesis (29). When commercial N’-methylnicotinamide is pur.ified to contain only 3 % of its original silver content, the inhibition is correspondingly diminished ; the remaining ‘Lirreversible” inhibition depends on enzyme concentration (Zone B behavior), and is directly proportional to the low concentrations of silver present, which could not be removed. It is not possible, therefore, to conclude from these data that N’-methylnicotinamide is an inhibitor of this enzyme. Inhibition in the presence of this reagent has been reported (10) ; 5.3 X 10M3M N’-methylnicotinamide resulted in 50 % inhibition in a reaction mixt*ure different from that used here, at pH 9.3, with an unspecified concentration of enzyme. This value is consistent with those in Fig. 1, using the contaminated commercial preparation and the highest enzyme concentration; a more direct comparison is not possible of course.

INHIBITION

171

The presence of silver in N’-methylnicotinamide also explains the concomitant decrease in the number of titratable sulfhydryl groups and in enzyme activity (Table II). This observation suggests caution not only in the interpretation of inhibition data obtained with this reagent, but also in the conclusion that it may be bound to sulfhydryl groups. A decrease in titratable -SH groups of yeast alcohol dehydrogenase on the addition of DPN and DPKH [but see (6, 7)] has similarly suggested that the coenzyme is bound to the enzyme through sulfhydryl groups (3, 5). It would seem desirable to eliminate contamination with metals as a possible basis for such observations, particularly since earlier methods of preparation of this coenzyme included multiple additions of basic lead acetat,e and silver nitrate (30). The findings with commercial semicarbazide indicate that a type of advenbitious inhibition may occur which is more subtle than the simple inactivation brought about by contaminating silver ions in N’-methylnicotinamide. Semicarbazide produces a timeand enzyme concentration-dependent inhibition even when there is a lo6 molar excess of the compound. On the basis of kinetic criteria (27, 28), semicarbazide itself cannot, be the actual inhibitor, and the absence of inhibition with the purified compound, in which metals are not detected, confirms this experimentally, The correlation between the loss of activity and the decrease in titratable enzyme sulfhydryl groups, and t’he arrest of the progressive inactivation upon the addition of noninhibitory concentrations of metal-binding agents, point to the action of metal ions. However, ferrous and cupric salts alone, in concentrations ident’ical to the amounts contaminating semicarbazide, do not inhibit the enzyme. Instead, a highly dissociable metal-semicarbazide or metal-urea complex might be postulated to be the act,ual inhibitor; such ligand-metal complexes are well known to inactivate enzymes (31), and their high affinity for active enzyme sites could account for an enzyme concentration dependence. It might be proposed alternatively that these synergistic inhibitions are due to phys-

172

HOCH,

MARTIN,

WACKER

ical changes in the struct’ure of the protein molecule, effectled by the organic reagents, since urea may he substituted for semicarbazide to enhance the inhibitory action of copper. Ahhough activity is not, altered by the organic agents directly, they might slowly make accessible the sites at which the metal ions bind to cause inhibition. These sites appear to be Ag+-titratable -SH groups (Fig. 5), as is seen with commercial N’-met,hylnicotinamide (Table II). The observed accelerations of the first-order rates of inact’ivation after enzyme and commercial semicarbazide have been in contact for periods of 40 min. or more (Fig. 2) may then represent such late changes in protein structure making -SH groups more available to Cu++ ions. Whatever t)he mechanism of the inhibit’ion by semicarbazide, the demonstration that the degree of inhibition depends on enzyme concentration serves to suggest it is adventitious w&h reference to the supposed organic inhibitor. The kinetic anomaly of an inhibition which depends on the concentration of the enzyme, when t’he concentration of the apparent inhibitor is in very large molar excess, can be used as a qualitative indication that an adventitious inhibition may exist. Obviously, act#ivity measurements do not substitut)e for analyt,ical demonstrations of contaminants, but the present data suggest, that the observation of enzyme concentration-dependent inhibition can serve as a valuable guide t’o adventitious inhibitions. REFERENCES 1. WAGNER-JAUREGG, T., AND MOELLER, E. F., 2. physiol. Chem. 236, 222 (1935). 2. NEGELEIN, E., AND WULFF, H.-J., Biochem. Z. 293, 351 (1937). 3. WALLENFELS, K., AND SUND, H., Biochem. Z. 329, 17 (1957). 4. DIXON, M., Suture 140, 806 (1937). 5. BARRON, E. S. G., AND LEVINE, S., Arch. Biothem. Biophys. 41, 175 (1952). 6. HOCH, F. L., AND ZOTOS, B., Federation Proc. 16, 359 (1957).

AND

VALLEE

7. HOCH, F. L., AND VALI~EE, B. L., in “Sulfur in Proteins” (R. Benesch et al., eds.), p. 245. Academic Press, New York, 1959. 8. VALLEE, B. L., AND HOCH, F. L., Proc. Katl. Acad. Sci. U. S. 41, 327 (1955). 9. KAPLAN, N. O., CIOTTI, M. M., AND STOLZENBACH, F. E., J. Riol. Chem. 221, 833 (1956). 10. VAN EYS, J., AND KAPLAN, X. O., Biochim. et Biophys. Acta 23, 574 (1957). 11. WALLENFELS, K., AND SUND, H., Biochem. Z. 329, 48 (1957). 12. VAN EYS, J., CIOTTI, M. M., AND KAPLAN, N. O., Biochim. et Biophys. Acta 23, 581 (1957). 13. VAN EYS, J., SAN PIETRO, A., AND KAPLAN, N. 0.) Science 127, 1443 (1958). 14. WALLENFELS, K., AND SUND, H., Biochem. Z. 329, 59 (1957). 15. REDETZKI, H. E., AND NOWINSKI, W. W., Nature 1’79, 1018 (1957). 16. VAN EYS, J., CIOTTI, M. M., AND KAPLAN, N. O., J. Biol. Chem. 231, 571 (1958). 17. WACKER, W. E. C., VALLEE, B. L., AND HOCH, F. L., Intern. Congr. Biochem., ZVth Congr., Vienna, 1958, Sec. 5, p. 57. 18. RACKER, E., J. Biol. Chem. 184, 313 (1950). 19. BALL, E. G., Biol. Bull. 64,277 (1933). 20. BENESCH, R. E., LARDY, H. A. AND BENESCH, R., J. Biol. Chem. 216, 663 (1955). 21. HOCH, F. L., WILLIAMS, R. J. P., AND VALLEE, B. L., J. Biol. Chem. 232, 453 (1958). 22. WILLIAMS, R. J. P., HOCH, F. L., AND VALLEE, B. L., J. Biol. Chem. 232,465 (1958). 23. VALLEE, B. L., Advances in Protein Chem. 10, 317 (1955). 24. VALLEE, B. L. AND HOCH, F. L., Intern. Rev. cyto1. 8, 345 (1959). 25. SANDELL, E. B., “Calorimetric Determination of Traces of Metals, 3rd ed., p. 842. Interscience Publ., New York, 1959. 26. ACKERMAN, W. W., AND POTTER, V. R., Proc. Sot. Exptl. Biol. Med. 72, 1 (1949). 27. STRAUS, 0. H., AND GOLDSTEIN, A., J. Gen. Physiol. 26,559 (1943). 28. GOLDSTEIN, A., J. Gen. Physiol. 27, 529 (1944). 29. KARRER, P., SCHWARZENBACH, G., BENZ, F., AND SOLMSSEN, U., Helv. Chim. Acta 19. 811 (1936). 30. LEPAGE, G. A., Biochem. Preparations 1, 28 (1949). 31. ALBFIRT. A., hTature 172, 201 (1953).