Selective binding of fluorescein mercuric acetate to yeast alcohol dehydrogenase

ARCHIVES

OF BIOCHEMISTRY

Selective

AND

127, 637-644 (1966)

BIOPHYSICS

Binding Yeast

of Fluorescein Alcohol

JAMES R. HEITZ Department

of Biochemistry,

Mercuric

Acetate

to

Dehydrogenasel BRUCE M. ANDERSON

AND

of Tennessee, Knoxuille,

University

Tennessee 37916

Received March 26, 1968; accepted May 15, 1968 Yeast alcohol dehydrogenase is rapidly inactivated by low concentrations of fluorescein mercuric acetate. This inactivation is accompanied by disaggregation of the enzyme to smaller molecular weight fragments. Incubation of the enzyme with a concentration of Auorescein mercuric acetate representing 3 moles of the mercurial per enzyme active site results in 85”< inactivation of the enzyme within 2 minutes at O-4”. After extensive dialysis of the inactivated enzyme, 2 moles of the mercurial per enzyme active site remain tightly bound to the enzyme. The binding of 2 moles of fluorescein mercuric acetate per enzyme active site was also demonstrated through spectrophotometric and fluorescence quenching techniques. Inactivation of the enzyme by this mercurial is partially reversed by the addition of cysteine. Prior inactivation of the enzyme by iodoacetic acid or iodoacetamide previously shown to block one sulfbydryl group per active site results in a modified enzyme which, on titration, will accept only 1 mole of fluorescein mercuric acetate per enzyme active site.

Nonpolar interactions have been demonstrated to contribute to the binding of coenzyme-competitive inhibitors to yeast alcohol dehydrogenase (1-6). The hydrophobic region of the enzyme involved in these interactions is thought to be located close to that part of the NAD binding site of the enzyme which interacts with the pyridinium ring system of the oxidized coenzyme (2, 4). Many of the coenzymecompetitive inhibitors that interact with the hydrophobic region of the enzyme can be bound simultaneously with coenzymecompetitive adenine derivatives that interact with a different region of the NAD binding site (4, 6-8). Previous considerations (9, 10) of the properties of the NAD binding site of yeast alcohol dehydrogenase suggested the importance of a sulfhydry1 group in the coenzyme binding process. In this respect, an investigation of the role of nonpolar interactions in the functioning of sulfhydryl reagents with yeast ’ Contribution No. 54 from the Department of Biochemistry, The University of Tennessee, Knoxville, Tennessee.

alcohol dehydrogenase was initiated. In a recent study with N-alkylmaleimides, nonpolar effects were observed in the inactivation of yeast alcohol dehydrogenase by these compounds (11). The lack of protection against maleimide inactivation of the enzyme by compounds bound at the pyridinium ring region of the NAD binding site, raises questions concerning the location of the sulfhydryl group involved. It was of interest, therefore, to extend this investigation to the study of inactivation of the enzyme by nonpolar mercurials. Yeast alcohol dehydrogenase has been demonstrated to be rapidly inactivated by p-hydroxymercuribenzoate (12, 13). The very effective binding of polycyclic nitrogen bases as coenzyme-competitive inhibitors of the enzyme (5) suggested the possibility that a polycyclic mercurial might likewise by very effective in the inactivation of the enzyme. Karush et al. (14), recently reported the synthesis of a polycyclic mercurial, fluorescein mercuric acetate (FMA)’ which was used as a rea’ FMA, fluorescein mercuric acetate.

637

HEITZ

AND

ANDERSON

gent to study disulfide linkages in proteins. The quenching of fluorescence of FMA on reaction with sulfhydryl groups was demonstrated in these studies. The chemical, spectral and fluorescence properties of FMA indicated several possibilities for the use of this mercurial to study the NAD binding site of yeast alcohol dehydrogenase. The proposed location of the essential sulfhydryl groups in a nonpolar region of the enzyme (11) suggested that inactivation by FMA should proceed through a very selective process. EXPERIMENTAL

PROCEDURE

Twice crystallized and one lyophilized yeast alcohol dehydrogenase (alcohol : NAD oxidoreductase, EC 1.1.1.1) was obtained from the Worthington Biochemical Corporation. Stock solutions of the enzyme were prepared fresh daily in 0.1 M sodium pyrophosphate buffer, pH 8.0. Fluorescein mercuric acetate was purchased from Nutritional Biochemicals Corporation. 2,7-Dichlorofluorescein and N-ethylmaleimide were products of Eastman Organic Chemicals. NAD, iodoacetic acid, iodoacetamide, p-hydroxymercuribenzoate, and L-cysteine hydrochloride were obtained from the Sigma Chemical Company. Sephadex G-200 and Blue Dextran were products of Pharmacia. Yeast alcohol dehydrogenase activity was measured as a function of the change in absorbance at 340 mp per minute in 3-ml standard assay mixtures containing 0.04 M sodium pyrophosphate buffer, pH 8.0, 0.1 M ethanol, and 3.0 X lo-” M NAD. The spectrophotometric assays were performed at 25’ in a Gilford Model 2000 Recording Spectrophotometer equipped with a temperature-controlled cell compartment or a similarly equipped Zeiss PMQ II Spectrophotometer. Visible and ultraviolet spectra were measured on a Bausch and Lomb Spectronic 505. Measurements of pH were made at 25” with a Radiometer pH Meter, type PHM 4b with a G-200-B glass electrode. Fluorescence spectra were measured on an Aminco-Bowman Spectrophotofluorimeter with a Xenon Mercury Lamp, Pacific Photometric Recording Photometer Model 15 fitted with an EM1 9502 Photocell, and a Moseley Autograf Model 135A X-Y Recorder. RESULTS

Yeast alcohol dehydrogenase was demonstrated to be rapidly inactivated by low concentrations of FMA. The time dependence of inactivation by this mercurial was studied in lo-ml incubation mixtures at

25

3 1

olki 0

3 ’ ’ ’ 20 ’ * ’ 30 ’ IO Time of incubation

1 40 ’ b’

L50 1

(minutes)

FIG. 1. Time dependence of yeast alcohol dehydrogenase inactivation with increasing concentrations of FMA. Each incubation mixture contained 3.75 X lo-” M yeast alcohol dehydrogenase in 0.1 M sodium pyrophosphate buffer at pH 8.0 and the following concentrations of FMA: line 1, none; line 2, 1.44 X 10-j M; line 3, 2.88 X lo-‘M; line 4, 4.32 X lo-‘M. The time of addition of enzyme was regarded as time zero. Enzyme activity was measured by transferring O.l-ml aliquots of the incubation mixtures to a standard yeast alcohol dehydrogenase assay mixture.

0.6 I

05

j

0.4

4

0.3 0.2 0.1 0 450

475

500

525

550

Wavelength (mpl

FIG. 2. Spectral changes of FMA upon addition of yeast alcohol dehydrogenase. Each reaction mixture contained 1.11 X 10 ~’ M FMA in 0.1 M sodium pyrophosphate buffer at pH 8.0 and the following concentrations of yeast alcohol dehydrogenase: line 1, none; line 2, 3.34 X lo-'M; line 3, 6.68 X 10m'M; line 4, 1 X 10m6M; line 5, 1.34 X lo-"M; line 6, 1.67 X lo-"M.

ice temperatures. The enzyme was incubated with different concentrations of inhibitor in 0.1 M sodium pyrophosphate, pH 8.0, and at various times, O.l-ml aliquots were transferred to standard assay mixtures to determine residual activity. The effect of FMA on yeast alcohol de-

FLUORESCEIN

MERCURIC

hydrogenase activity is shown in Fig. 1. Over the course of 50 minutes, the enzymatic activity in the absence of inhibitor (line 1) showed no measurable decrease. When the enzyme was incubated with 1.44 X lo-’ M FMA, a concentration which can also be expressed as a 1: 1 ratio of FMA molecules to enzyme active sites, inhibition of the enzyme was ob60rr served within 2 minutes. At 2: 1 and 3: 1 ratios of FMA to active sites, 85 and 97!‘C respectively, were obtained inhibition, within 2 minutes. Since polycyclic nitrogen bases are known to effectively inhibit yeast alcohol dehydrogenase (5)) inactivation by the fluorescein nucleus was investigated by incubating the enzyme with 2,7-dichlorofluorescein under the conditions described above. At the concentrations used with FMA, no inactivation by 2,7-dichlorofluorescein was observed. At a concentration of 1. X 10-j M 2,7-dichlorofluorescein (a 65: 1 ratio with respect to active sites) ap-

proximately 15:; inhibition of the enzyme was obtained. Higher concentrations of 2,7-dichlorofluorescein were not studied due to their interference with the spectrophotometric assay of the enzyme. A slight protection effect by NAD was observed in the inactivation of yeast alcohol dehydrogenase by FMA. Inactivation of the enzyme by 2.88 X 10-j M FMA which reached 85’; in 2 minutes, in the presence of 1.41 X 10 -’ M NAD required approximately 7 minutes to reach the same extent of inactivation. Also, if the enzyme is allowed to reach 85CC inactivation with 2.88 X 10mTM FMA, the addition of 1 X lo-“ M cysteine at this time (2 minutes) partially restores the enzyme activity to approximately 60% inactivation. When yeast alcohol dehydrogenase was added to FMA, a change in the FMA spectrum was observed. The effect of increasing yeast alcohol dehydrogenase concentration on the FMA spectrum is shown in Fig. 2. Free FMA has a molar extinction

TABLE THE

BINIIING

OF

FhIA

639

ACETATE

TO YEAST

I ALCOHOL

DEHYDROGENASE

Reaction mixtures (10 ml) were prepared containing 4.15 X 10-j JI yeast alcohol dehydrogenase in 0.1 1~ sodium pyrophosphate buffer, pH 8.0 atld the three concentrations of FMA listed. The concentration of the ellxyme espressrd ill terms of active sites was 1.66 X 1OF M. After 18 hours dialysis at O-4” against three changes of 0.1 31 sodium pyrophosphate buffer, pH 8.0, the ratio of FhfA per active site was determined by sprc~trophotometric assay. The 508.nip absorbance of the dialyzate was used as a measure of the FRIA rollcelltration. The contrihrltion to the 280 mp absorbance of that roluzent,rat,ion of FMA was subtracted from the observed absorbanre at 280 m@, to obtain the 280 absorbanre due to the protein alone. The extinction coefficient of 1.88 X 1Oj detcrmitred by Hayes and \-click (15) for t,he enzyme was llsed to ralculate the concentration of enzyme active sites. Dialysis stltdies with cysteine xere performed in the same manner with the cysteine added to the pyrophosphnte buffer.

Before

Ratio 2

Ratio 3

1 (Xi X 1OP 1.68 X 1W 6 1:1

X

4.94 x IO-” 3: 1

1 .(i(i X 10P 8.30 x 10-e 5:l

Dialysis

Yeast alcohol dehydrogellase FhlA (.M) FMA: active sites

.Iflfu

Ratio 1

active

sites (>\I)

1 .Mi

1OP

diatysis

Yeast alcohol dehydrogerrasc FhlA (M) FhlA: active sit,es

active

sites (11)

.tfter dialysis again.71 8 X 10m3.v c!ysleinc Yeast alcohol dehydrogennse active sites FMA (M) FhlA: active sites

(XI)

2.78 x 10 6 9.01 x IO-’ 0.32:1

1.82 3.81

2.09:1

2.03 X 1OP 4.06 x 10-C 2.00:1

2.18 X 1OF 4.17 X 10-j 0.17:1

2.91 x 10-6 1.12 X 10-G 0.39:1

3.85 x lo-‘; 1.92 X 10-G 0.5O:l

x x

10-G 10-C

640

HEITZ

AND

ANDERSON

it was of interest to determine if the same phenomenon occurred upon reaction of t this compound with yeast alcohol dehy60 drogenase. The addition of yeast alcohol t 70 dehydrogenase to a solution of FMA in 0.1 2 M sodium pyrophosphate buffer, pH 8.0 f: 60resulted in a rapid decrease in the FMA g 50fluorescence (Fig. 3). On the basis of this 0 quenching of fluorescence, the enzyme was 2 403 titrated with FMA. The fluorescence of lL 30fourteen concentrations of FMA was measured alone and in the presence of three 20t different concentrations of yeast alcohol dehydrogenase. The fluorescence intensities obtained, plotted according to Hsu 450 500 550 600 600 and Lardy (16), are shown in Fig. 4. The Wovelength (mp) amount of FMA interacting with the difFIG. 3. Fluorescence emission spectra of FMA ferent concentrations of enzyme was caland of FMA with added yeast alcohol dehydrogenculated from the points of intersection ase. Line 1, 3.1 X lo-'M FMA in 0.1 M sodium pyshown for lines 2, 3, and 4. The average rophosphate buffer at pH 8.0. Line 2, 3.1 X lo-‘M value calculated from the three titration FMA and 2 X 10m7 M yeast alcohol dehydrogenase curves indicates 2.08 molecules of FMA in 0.1 M sodium pyrophosphate buffer at pH 8.0. bound per active site of the enzyme. The solutions were excited at 495 mr. Fluorescence titrations were also percoefficient of 7.80 x lo4 at 499 rnF (14). At formed by holding the FMA concentration a ratio of approximately 2: 1 FMA to en- constant and increasing the yeast alcohol zyme active sites, the maximum spectral dehydrogenase concentration. Ten concenchange was obtained, resulting in a new trations of enzyme were added to three molar extinction coefficient of 4.78 X lo4 different concentrations of FMA. The at 508 rnp. The molar extinction coeffi- change in fluorescence intensity on addicient of FMA at 280 rnp of 9.31 X 103, tion of enzyme is shown in Fig. 5. The condid not change significantly upon titration with enzyme. The tightness of binding of FMA to 200 yeast alcohol dehydrogenase was studied through dialysis experiments and the results of these studies are shown in Table I. After incubation of the enzyme with excess FMA, followed by dialysis, two FMA molecules per enzyme active site were found tightly bound to the enzyme. FMA-inactivated yeast alcohol dehydrogenase was also dialyzed against 2 X lo-” 2.0 _ 3.0 0 1.0 Fluorescein mercuric acetate (MxlO’) M cysteine in 0.1 M sodium pyrophosphate buffer, pH 8.0. After 18 hours dialysis, FIG. 4. Fluorescence titration of yeast alcohol dethe amount of FMA remaining bound to hydrogenase with FMA. Reaction mixtures contained the enzyme was considerably less than 0.1 M sodium pyrophosphate buffer at pH 8.0 and that observed after dialysis in the absence the following concentrations of yeast alcohol dehyof cysteine (Table I). drogenase: line 1, none; line 2, 4.78 X 10m9 M; line 3, Since Karush et al. (14) had shown that 9.26 X 1om9M; line 4, 1.34 X 10m8M. The solutions the fluorescence of FMA decreased upon were excited at 495 mp and emission was measured reaction with various sulfhydryl groups, at 525 mr. so

YjikLxJ

FLUORESCEIN

0

0.5

1.0

Yeast alcohol

1.5 dshydrogsnoss

2.0

MERCURIC

2.5

(Mx108)

FIG. 5. Fluorescence titration of FMA with yeast alcohol dehydrogenase. Reaction mixtures contained 0.1 M sodium pyrophosphate buffer at pH 8.0 and the following concentrations of FMA: line 1, 3.65 X ~O-“M; line 2, 7.06 X IO-“M; line 3, 1.03 X ~O-‘M. The solutions were excited at 495’mp and emission was measured at 525 rnp.

centrations of yeast alcohol dehydrogenase required to titrate the different amounts of FMA present can be obtained from the points of intersection and the average of the three values calculated, indicated 1.92 molecules of FMA bound per active site of the enzyme. The fluorescence titration of yeast alcohol dehydrogenase with FMA was also carried out after the enzyme had been greater than 95”; inactivated by various sulfhydryl reagents. For example, three concentrations of yeast alcohol dehydrogenase inactivated by N-ethylmaleimide were titrated with FMA. No appreciable interaction of FMA with the inactivated enzyme was observed in the FMA concentration range previously shown to titrate the native enzyme, indicating no binding of FMA to the N-ethylmaleimideinactivated enzyme. Yeast alcohol dehydrogenase inactivated with iodoacetic acid or iodoacetamide was observed through fluorescence titration studies to bind one molecule of FMA per active site of the enzyme. Yeast alcohol dehydrogenase inactivated with p-hydroxymercuribenzoate was observed to bind two molecules of FMA per active site. The possibility that protein structural changes occur on the inactivation of yeast alcohol dehydrogenase by FMA was investigated using density gradient centrif-

641

ACETATE

ugation and gel filtration procedures. Sucrose density gradients were prepared on a device described by Salo and Kouns (17). Sucrose solutions of 5 and 20c; (w/v) buffer, in 0.1 M sodium pyrophosphate pH 8.0, were used to prepare 4.5-ml linear gradients in cellulose nitrate tubes. The gradients were run for 18 hours at 39,000 revolutions per minute in a SW 39 head on a Sorvall RC-2 refrigerated centrifuge. The fractions were collected in seven-drop units by means of a tube puncturing device described by Salo (18). The results of sucrose density gradient centrifugation of yeast alcohol dehydrogenase and of FMA-inactivated yeast alcohol dehydrogenase are shown in Fig. 6. The FMA-containing material was separated from the active enzyme by 14 tubes in the collection procedure. Protein was present in both peaks. Gel filtration studies were carried out with Sephadex G-200 prepared in 0.1 M sodium pyrophosphate buffer, pH 8.0. The column used was 11 mm in diameter, 52 mm in height and contained 47.7 ml of gel bed volume. The flow rate of the column was approximately 6 ml per hour. Figure 7 shows the results of the Sephadex G-200 separation of several proteins. Peak 1 represents the void volume (16.1

-0

7

14

21

28

35

42

49

Tube number

FIG. 6. Sucrose density gradient centrifugation of active yeast alcohol dehydrogenase and FMA-inactivated yeast alcohol dehydrogenase. Line 1, active yeast alcohol dehydrogenase measured by enzymatic assay. Line 2, FMA measured by 508-rnr assay. Line 3, FMA-inactivated yeast alcohol dehydrogenase measured by 280-mp assay.

642

HEITZ

AND

ANDERSON

are consistent with the inactivation of the enzyme occurring through the binding of FMA to sulfhydryl groups of the enzyme. Yeast alcohol dehydrogenase inactivated by p-hydroxymercuribenzoate was previously demonstrated to be reactivated by glutathione (13). Although inhibition of yeast alcohol dehydrogenase by polycyclic nitrogen bases alone has been observed (5), this type of inhibition is normally reversible and requires much higher concentrations of the inhibitor. In this Elulion volume (ml 1 respect, only 15% inhibition of yeast alcohol dehydrogenase was observed with FIG. 7. Sephadex G-200 elution pattern. Peak 1, 1 X 10m5M 2,7-dichlorofluorescein which Blue Dextran. Peak 2, active yeast alcohol dehydrohas properties similar to those of the genase. Peak 3, duplicate determinations of FMA-influorescein nucleus of FMA. activated yeast alcohol dehydrogenase. Peak 4, huThe rapid inactivation of yeast alcohol man hemoglobin. Peak 5, glycylglycine. dehydrogenase by FMA can be correlated ml) of the column as determined by exclu- with the binding of two moles of the sion of Blue Dextran measured at 620 rnp. mercurial per active site of the enzyme. Peak 2 represents active yeast alcohol de- Analysis of the inactivated enzyme after hydrogenase, elution volume 24.7 ml, extensive dialysis revealed the presence which was determined by standard en- of two moles of FMA per enzyme active zyme assay. Peak 3 represents two inde- site; however, much of this tightly bound pendent determinations of FMA-modified FMA could be removed by dialysis against yeast alcohol dehydrogenase. These two cysteine (Table I). This ratio of 2: 1, FMA determinations were made on separate en- to enzyme active sites, was also calculated zyme preparations chromatographed on from the spectral changes obtained on the different days. This inactive enzyme form addition of yeast alcohol dehydrogenase to was assayed at 508 rnp and the elution FMA (Fig. 2). Fluorescence titration exvolume was 30.1 ml. Peak 4 represents periments, performed as a function of inhuman hemoglobin, which was assayed by creasing concentrations of FMA (Fig. 4) or 415 rnp absorption and had an elution increasing concentrations of yeast alcohol volume of 32.3 ml. Peak 5 represents gly- dehydrogenase (Fig. 5), likewise indicated cylglycine (the salt front) and was meas- the binding of two moles of FMA per acured by 210 rnp absorption. The elution tive site of the enzyme. The low concentration of FMA required volume of the salt front was approximately to inactivate yeast alcohol dehydrogenase 42 ml. suggests the inactivation to be a very DISCUSSION specific and efficient process. Incubation Yeast alcohol dehydrogenase has been of the enzyme with a concentration of demonstrated to be inactivated rapidly at FMA representing a 3: 1 ratio of FMA to enzyme active sites, produces 85% inac0-4O by very low concentrations of FMA (Fig. 1). The inactivation observed was tivation of the enzyme in 2 minutes at not reversed by dilution into the assay O-4” and results in the tight binding of 2 medium or by extensive dialysis against moles of FMA per active site of the ensodium pyrophosphate buffer (Table I). zyme. Incubation at a ratio of 5: 1, FMA After attainment of 65% inactivation of to enzyme active sites, did not increase the enzyme, a partial restoration of en- the number of moles of FMA tightly zyme activity was achieved by the addi- bound to the enzyme (Table I). These data tion of 1 X 10e4 M cysteine. These data indicate the presence of eight sulfhydryl

FLUORESCEIN

MERCURIC

groups per mole of enzyme that can be titrated prior to any further reaction of FMA with other sulfhydryl groups of the enzyme. Yeast alcohol dehydrogenase has been reported to contain 38 cysteine residues per mole of enzyme (19). It is felt that inactivation of the enzyme does not require the blocking of all eight of the reactive sulfhydryl groups. Incubation of the enzyme with FMA at a ratio of 1: 1, FMA to active sites, routinely produced more than 50% inactivation of the enzyme (Fig. 1). Inactivation of yeast alcohol dehydrogenase by iodoacetic acid was previously correlated to the modification of one sulfhydryl group per active site of the enzyme (20, 21). Yeast alcohol dehydrogenase totally inactivated by iodoacetic acid or iodoacetamide was demonstrated through fluorescence quenching techniques to retain the ability to bind one mole of FMA per active site of the enzyme. It would appear that the blocking of one sulfhydryl group per active site would produce the inactive form of the enzyme. It is not presently clear why eight sulfhydryl groups of the enzyme should show similar reactivities towards the mercurial, FMA, but differ in reactivity towards iodoacetic acid and iodoacetamide. It is known that yeast alcohol dehydrogenase disaggregates to smaller molecular weight fragments on reaction with mercurials (13). The inactivation of yeast alcohol dehydrogenase by FMA is likewise accompanied by changes in protein structure (Figs. 6 and 7). It is conceivable that the inactivation of yeast alcohol dehydrogenase by FMA proceeds through the blocking of one sulfhydryl group per active site (a total of four), followed by the release of four more, equally reactive sulfhydryl groups on disaggregation of the enzyme. The molecular weight of FMA-inactivated yeast alcohol dehydrogenase was estimated from density gradient sedimentation studies (Fig. 6) to be 37,000 and from gel filtration studies (Fig. 7) to be 36,000, assuming no drastic conformational changes. Dissociation of yeast alcohol dehydrogenase into four subunits was previously observed as a result of the

643

ACETATE

effects of chelating agents (22), sodium dodecyl sulfate (23) and urea (24). Studies are currently in progress to characterize further the smaller molecular weight material produced by FMA inactivation of yeast alcohol dehydrogenase. The inactivation of yeast alcohol dehydrogenase by maleimides was recently demonstrated to be facilitated by nonpolar interactions between the inhibitors and the enzyme (11). The eight sulfhydryl groups of yeast alcohol dehydrogenase, readily titrated by FMA (Fig. 4), were observed not to be available for reaction with FMA in yeast alcohol dehydrogenase inactivated by N-ethylmaleimide. The nonpolar interactions that are of importance in the maleimide inactivation of the enzyme, may also play a role in the dissociation of the enzyme into subunits. Such a relationship would also be consistent with the dissociation of the enzyme by sodium dodecyl sulfa e and urea. ACKNOWLEDGMENTS This work was supported by Grant GB-5503 from the National Science Foundation. We wish to thank Dr. Jorge E. Churchich for valuable discussion of the fluorescence studies. REFERENCES 1. ANDERSON, B. M. AND ANDERSON, C. D., Biochem. Biophys. Res. Commun. 16, 258 (1964). 2. ANDERSON, B. M., REYNOLDS, M. L. AND ANDERSON, c. D., Biochim. Bionhys. Acta 99, 46 (1965). 3. ANDERSON, B. M. AND REYNOLDS, M. L., Biochim. Biophys. Acta 96, 45 (1965). 4. ANDERSON, B. M. AND REYNOLDS, M. L., Arch. Biochem. Biophys. 111, 1 (1965). 5. ANDERSON, B. M., REYNOLDS, M. L. AND ANDERSON, C. D., Biochim. Biophys. Acta 113, 235 (1966). 6. ANDERSON, B. M. AND REYNOLDS, M. L., Arch. Biochem. Biophys. 114, 299 (1966). I. FONDA, M. L. AND ANDERSON, B. M., Arch. Biothem. Biophys. 120,49 (1967). 8. ANDERSON, B. M., REYNOLDS, M. L. AND ANDERSON, C. D., Arch. Biochem. Biophys. 111, 202 (1966). 9. WALLENFELS, K. AND SUND, H., Biochem. 2. 329, 59 (1957). 10. VAN EYS, J. AND KAPLAN, X. O., Biochim. Biophys. Acta 23, 574 (1957).

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11. HEITZ, J. R., ANDERSON, C. D. AND ANDERSON, B. M., Arch. Biochem. Biophys. 127, 627-636 (1968). 12. HOCH, F. L. AND VALLEE, B. L., in R. Benesch et al. Sulfur in Proteins, p. 245. Academic Press, New York (1959). 13. SNOLXRASS,P. J., VALLEE, B. L. AND HOCH, F. L., J. Biol. Chen.235, 504 (1960). 14. KARUSH, F., KLINMAN, N. R. AND MARKS, R., Anal. Biochem. 9, 100 (1964). 15. HAYES, J. R., JR. AND VELICK, S. R., J. Biol. Chem.207,225( 1954). 16. Hsu, R. Y. AND LARDY, H. A., J. Biol. Chem. 242,527(1967).

ANDERSON 17. SAI.O, T. P. AND KOUNS, D. M., Ad. Biochem. 13, 74 (1965). 18. SALO, T. P., And. Biochem. 10,344 (1965). 19. WALLENFELS, K. AND ARENS, A., Biochem. 2. 332, 217, (1960). 20. HARRIS, I., Nature 203, 30 (1964). 21. WHITEHEAD, E. P. AND RABIN, B. R., Biochem. J., 90, 532 (1964). 22. KAJI, J. H. R. AND VALLEE, B. L., J. Biol. Chem. 235,3188 (1960). 23. HERSH, R. T., Biochim. Biophys. Acta 58, 353

(1962). 24. OHTA, T. AND OGURA, Y., J. Biochem. (Japan) 58, 73(1965).