Yeast Aldehyde Dehydrogenase

Vol.

THE JOURNAL 242, No. 21,

OF BIOLOGICAL

Issueof November Printed

Yeast

in

CHEMISTRY 10, PP. 5019-5023,

Aldehyde

I. PURIFICATION

1967

U.S.A.

AND

Dehydrogenase CRYSTALLIZATION (Received for publication,

July 24, 1967)

CHARLES R. STEINMAX* AND WILLIAM B. JAKOBY From the Section on Enzymes and Cellular Biochemistry, National Institute of Arthritis and Metabolic Diseases,National Institutes of Health, Bethesda,Maryland 20014

SUMMARY

Partly because of the wide distribution of aldehyde dehydrogenases and their substrates, a variety of such enzymes has been described (1). Despite the abundance of preparations catalyzing the pyridine nucleotide-linked oxidation of aldehydes to the corresponding carboxylic acids (I), no one enzyme has been purified to a state approaching homogeneity, thereby limiting the number of approaches to a study of mechanism. With the latter application in mind, it was decided to choose a relatively stable, easily available, and broadly specific aldehyde dehydrogenase, and to purify it to the appropriate degree. This report describes the purification, crystallization, and certain physical features of the potassium-dependent aldehyde dehydrogenase from yeast (EC 1.2.1.5) which was originally studied by Black (2, 3). MATERIALS

AND

METHODS

Fresh bakers’ yeast was obtained from a local distributor of the Anheuser-Busch Brewing Company and was used within 24 hours of purchase. All aldehydes were of the best grade commercially available and were used without further purification. Glycerol was a reagent grade product of the Fisher Scientific Company. Nucleotides were prepared by Sigma, potassium * Present address, Department Hospital, New York, New York.

of Medicine,

Presbyterian

EnzymeAssay The rate of formation of DPNH was followed spectrophotometrically at 340 rnp and 25” in a Beckman DU spectrophotometer with a Gilford automatic cuvette changer and Brown recorder. The following components were added in the order listed and were used at the noted concentrations in a total volume of 1.0 ml: 0.2 M KCl; 0.1 M Tris-chloride at pH 8.0; 1 mM DPN; 1 mu reduced glutathione; enzyme; 0.6 mu benzaldehyde. Since there may be a lag in the appearance of DPNH during the initial 1 to 2 min, the maximal linear rate was taken as the assay value. Under these conditions the reaction was linearly proportional to enzyme concentration when absorbance changes of less than 0.250 per min were obtained. An absorbance change of 0.001 per min may be measured readily when the sensitivity of the Gilford (model 2000) recordingspectrophotometer is increased so that a full scale deflection on the recorder represents an ab-

5019

Downloaded from www.jbc.org by guest, on October 17, 2011

The potassium-activated, pyridine nucleotide-linked aldehyde dehydrogenase from yeast has been purified to the stage of homogeneity as judged by ultracentrifugation and gel electrophoresis. The enzyme has been crystallized, although this is not a recommended step in purification because loss of catalytic activity is thereby incurred. At least three separable, active fractions were obtained with the large-scale purification procedure presented in this report. However, only one major fraction was found when precautions were taken to minimize proteolysis. The purified aldehyde dehydrogenase has a molecular weight of 200,000.

glutathione by Schwarz, and phenylmethylsulfonylfluoride by Calbiochem. All reagents for disk gel electrophoresis, using the method of Ornstein (4) at pH 9.5, were supplied by Canalco. DEAE-Cellulose (Schleicher and Schuell, type 70) was washed six times, using 400 volumes each, with water over a 2-day period; fine particles were discarded. The sediment was washed twice with 100 volumes of the same buffer used for chromatography. After the column was poured, 5 volumes of the appropriate buffer were passed through it before charging with protein. DEAE-Sephadex (Pharmacia, A-50) was hydrated for 1 The gel was washed with an addiday in 3 volumes of water. tional 5 volumes of water and then with 3 volumes of the same buffer used for chromatography. After storage in buffer for one day, the gel was washed with another 3 volumes of buffer and stored until used. Subsequent to forming a column, the gel was washed with 3 column volumes of buffer before charging with protein. Elution from ion-exchange columns was monitored by determination of both activity and conductivity; the latter measurement was performed at room temperature with a Radiometer conductivity meter. Some variability was encountered in the conductivity at which a particular protein was eluted, although a given ion-exchange resin, prepared in an identical fashion, yielded a reproducible elution pattern.

5020

Yeast Aldehyde TABLE I Summary of purification

Dehydrogenase.

I

Vol.

242, n’o. 21

Enzyme Purification procedure

The procedure applies to 25.pound lots of yeast and yields approximately 50 mg of homogeneous protein after concentration of a DEAE-cellulose eluate fraction. The results of one preparaml units u?zits/mg tion are summarized in Table I. w Yeast was crumbled by hand and frozen by gradual addition to -0 1 Extraction and 4-liter beakers containing liquid nitrogen as described by Black heating (3). As the nitrogen evaporated, it was replaced until approx2 Pooled acid 640 4020 5100 0.78 eluates imately 4 pounds of frozen yeast had accumulated in each of 6 Protamine 3 720 4900 5030 0.92 beakers. The vessels were set aside at 4” and the residual treated nitrogen was allowed to evaporate. To each beaker were added 4 Ammonium sul78 2900 1620 1.8 1500 ml of 0.3 M K2HP04 and, after initial mixing, 15 ml of a fate solution of phenylmethylsulfonylfluoride (16 mg per ml of n5A DEAE-cellu5100 352”~~ 51 7.0 propanol)l were added dropwise with stirring. The mixture was lose, Peak A& allowed to remain at 4” for approximately 20 hours, by which 5B DEAE-Sepha1400 707”z 0 101 7.0 time thawing was almost complete. Any remaining masses of dex, Peak Ab frozen yeast were dispersed and an additional 15 ml of the 0 Because of contamination by alcohol dehydrogenase and its phenylmethylsulfonylfluoride reagent were added to each beaker. substrate, the activity of this fraction could not be assayed by the The mixture was stirred for 20 hours and the pH was subsespectrophotometric method used. quently adjusted to 5.5 by the slow addition of 2 N HCI. b Only 10 to 20% of this fraction was used for the chromatogHeat Step-The mixture was heated in a 50-liter water bath raphy step. The amounts used for the preparations summarized Batches of 1.8 liters in 2-liter beakers were maintained at 60”. here are noted in the text. stirred vigorously for 15 min and were rapidly cooled thereafter c The yield of activity is, of course, dependent on the choice in a salt-ice bath. The sediment was discarded after centrifugaof fractions representing Peak A. This information is presented tion for 10 min at approximately 4” and 5000 x g, and all subsein Fig. 1 for the preparation described. quent procedures were carried out at this temperature. Acid Treatment-The supernatant fluid was adjusted to pH 5.1 sorbance of 0.100. One unit of activity is defined as that amount with 1 M citric acid. After stirring for 30 min, the precipitate of enzyme producing 1.0 pmole of DPNH per min under the described conditions and is equal to an absorbance change of formed by this treatment was separated with a Sorvall continuous flow centrifuge and discarded. Citric acid, 1 M, was added to pH 6.21 at 340 rnp. The rate of DPNH formation with 0.17 mM 4.3. The precipitate was collected by continuous flow centrifugaacetaldehyde and otherwise identical assay conditions was greater tion and extracted by suspension in approximately 150 ml of 0.1 by a factor of 6.7 when compared to benzaldehyde M potassium phosphate buffer at pH 6.5 containing 50 mM thioglycerol. A rapidly rotating, cylindrical, glass homogenizer Protein Concentration fitting the 50-ml metal centrifuge cups of the Servall centrifuge Specific activity is expressed as units per mg of protein. Prowas convenient for this purpose. After homogenization, the pH tein was estimated by the method of Lowry et al. (5) for all was adjusted to 8.0 with 1 N KOH, the suspension was stirred for preparations prior to the stage of DEAE-cellulose chromatog5 min, and the resultant slurry was centrifuged. The pH of the raphy. Subsequent preparations were assayed spectrophotosupernatant fluid was then lowered to 6.5 with 0.5 M citric acid metrically (6) ; a control tube containing the same solvent as that and set aside. The residue was again homogenized with 150 ml in which the protein was dissolved was necessary since the of the same buffer. After homogenization, the pH of the susabsorbance of the solvent, at lower wave lengths, increased with pension was adjusted to 9.0 by the addition of 0.1 N KOH and time. One milligram of the enzyme, as determined by the the suspension was stirred for 5 min. The slurry was centrifuged method of Lowry et al. (5), had an absorbance of 0.67 at 230 rnp and the pH of the supernatant fluid was adjusted to 6.5 with in a l-cm light path and contained 120 pg of nitrogen. The 0.5 M citric acid and set aside. This extraction procedure was absorbance ratio at 280 rnp to 260 rnp was 1.7. repeated once or twice, depending upon the yield, and the supernatant fluids resulting from each of the three or four Hydrodynamic Studies extractions were pooled. A Spinco model E analytical instrument equipped with a Protamine Sulfate-To each 100 ml of pooled extract, 15 ml of phase plate as schlieren diaphragm and at a controlled rotor freshly prepared 1% protamine sulfate in water were added temperature of 25.0” was used for all sedimentation studies. slowly. The precipitate was removed by centrifugation and Sedimentation velocity experiments were conducted at 25.0” in discarded.2 single-sector cells, one of which was fitted with a wedge window. 1 The solution of the inhibitor in n-propanol was at approxiSedimentation constants were obtained at a rotor speed of 60,000 mately 25” before adding to the cold extract. rpm and the values calculated from such experiments in a buffer 2 Another grorip experienced a 50yo loss of activity in repeating the protamine step; specific activity was not affected. Since solution were corrected to water at 25”. The diffusion coefficient prepara,tions do differ, it may be necessary to try was determined in the ultracentrifuge at 5000 rpm with the use commercial this step on a pilot scale before committing the entire preparation. of a double-sector, synthetic boundary cell and Rayleigh optics. Alternatively, we have omitted the step entirely in relatively Viscosity was determined with an Ubbelohde viscosimeter and small scale prepa.rations without apparent adverse effect on density with a calibrated pycnometer. subsequent manipulations.

-

NO.

-

Fraction

VdllIlle

Total activity

Total protein

Specific activity

Downloaded from www.jbc.org by guest, on October 17, 2011

Issue of November

10, 1967

C. R. Xteinman

3 The phosphate-Tris buffer was prepared from a stock solution of 0.05 M Tris; 0.1 M KHpPOh, and 5 GMkthylenediaminetetraacetic acid adiusted to DH 8.0 with N KOH at 4”. To 100 ml of this solution were addkd 250 ml of glycerol, 5 ml of thioglycerol, and sufficient water to bring the volume to 1 liter; the pH of the final buffer solution was between 7.5 and 7.6 at 4”.

0 0.E i

5021 100

200

300 I

DEAE-CELLUL’OSE )/-

I

_--

0.4 I-

0.2

1c/

I

DEAE-SEPHADEX

0.4

0.2 I

0

01:

cc

t

50

I

I

100

150

FRACTION

FIG. 1. Elution patterns of aldehyde dehydrogenase activity from DEAE-cellulose (tog) and DEAE-Senhadex (bottom) obtained from the prepara& described in the’ text. I-, en&me activity; - - -, conductivity at room temperature. The broad bars delineate those fractions chosen to represent a particular peak of activity.

Table I and Fig. ., selected fractions were individually concentratzd by ultrafiltration with a Scheicher and Schuell apparatus and were examined by disk gel electrophoresis. Only one protein band was found to be present in Fractions 122, 136, 146, 152, and 160 obt,ained from DEAE-cellulose (Fig. l), and, within limits of the method, the protein band of each of these fractions migrated at an identical rate. It was only at Fraction 170 and later eluates that more than one band appeared. Similarly, Peaks A and 13, as obtained by DEAE-Sephadex chromatography, yielded single but separate protein bands. In each case, the desired fractions were pooled, brought to 0.1 M with respect to potassium phosphate at pH 6.5, and concentrated by ultrafiltration. When the A protein was subjected to chromatography on a column of Sephadex G-200 (1.5 X 73), a single, coincident curve was obtained for both enzyme activity and protein concentration. After ultrafiltration the enzyme was stored at -90”. Enzyme activity of such preparations remained constant during storage for 2 months at -90” or for 1 day at room temperature. However, repeated freezing and thawing led to inactivation and it is suggested that the preparation be stored in small aliquots to be thawed as used. Preparations of Peaks A and B were free of both DPNH oxidase and alcohol dehydrogenase. Fresh additions of phenylmethylsulfonylfluoride had no effect on activity or on storage properties of either protein species. Crystallization-An equal volume of 0.1 M potassium phosphate

Downloaded from www.jbc.org by guest, on October 17, 2011

Salt Prec+itation-Solid ammonium sulfate, 25.2 g/100 ml, was gradually added with stirring and allowed to stand for 30 min. The precipitate was removed by centrifugation and discarded. An additional 9.8 g of ammonium sulfate were added to the supernatant fluid and, after 30 min, the precipitate was collected and dissolved in 100 ml of 0.1 M potassium phosphate buffer at pH 6.5 containing 50 mM thioglycerol, 0.25 mM ethylenediaminetetraacetic acid, and 0.1 ml of phenylmethylsulfonylfluoride (18 mg per ml in n-propanol). The resulting clear solution was rapidly frozen in 4-ml aliquots with liquid nitrogen, under which conditions it remained stable at -90” for 2 months. Different preparations varied in their ability to retain activity beyond this period. In particular, slow or repeated freezing and thawing resulted in inactivation. DEAE-cellulose-A portion of the ammonium sulfate fraction containing 350 units of enzyme was desalted by passage through a column of Bio-Gel P-20 (2.5 x 15 cm) which had been previously equilibrated with a phosphate-Tris buffer3 containing 0.03 M KCl. Alternatively, the preparation was dialyzed overnight against 100 volumes of the same buffer. The protein solution was applied to a column of DEAE-cellulose, 2.5 x 75 cm, and eluted with a linear gradient. The mixing flask contained 4 liters of the phosphate-Tris buffer, made 0.03 M with respect to KCl. The reservoir contained 4 liters of the same buffer in 0.12 M KCl. Fractions of 22 ml each were collected at a flow rate of approximately 250 ml per hour. The elution pattern is presented in Fig. 1. It is apparent that this procedure separates several species of aldehyde dehydrogenase, a result which was consistently obtained. Indeed, the salt concentration of the eluting buffer was chosen in order to separate these protein species. The area of the curve in Fig. 1 labeled Peak A, comprising Fractions 122 to 142, was selected for further studies because it r-presented the largest single enzyme component. The total yield of eluted enzyme activity was approximately 60 % of that applied. DEAE-Sephadex-As an alternative to chromatography on DEAE-cellulose, DEAE-Sephadex was used with a somewhat clearer separation of catalytically active components A, B, and C (Fig. 1). The ammonium sulfate fraction (55 units of activity) was passed through Bio-Gel P-20 equilibrated with the phosphate-Tris buffer3 containing 0.05 M KC1 or was dialyzed against the same buffer as described for the DEAE-cellulose step. The resultant preparation was used to charge a DEAE-Sephadex column, 2.5 x 65 cm, previously equilibrated with the phosphateTris buffer supplemented to 0.05 M KCl. A linear gradient was applied with 700 ml of the buffer, 0.05 M with respect to KCl, in the mixing flask and 700 ml of the buffer, 0.25 M with respect to KCI, in a reservoir. The lower level of the two buffer containers was arranged so as to be at the same height as the initial top of the ion-exchange bed; DEAE-Sephadex contracts in volume over the range of ionic strength used. Fractions of 9 ml each were collected at a rate of approximately 10 ml per hour. The total yield of enzyme activity was equal to that obtained with DEAEcellulose. Concentration and Purity-In the preparation summarized in

and W. B. Jakoby

Yeast Aldehyde Dehydrogenase.

Vol. 242, No. 21

I PiAK

A

’ PEAK

b

’

.OE

0.2 .04

0.1

.02

2 5 a

0

2 z 3 .04

0

FIG.

initial

2. Dark field phot.omicrograph magnification of lOOO-fold. -----7---.

of enzyme crystals

--1-v

I

DEAE-SEPHADE Peak A

Peak

x/ 7

DEAE-CELLULOSE

Peak wm

at an

A

8

0.04

! 5 2

-i/

0.02; a :

/ T

*

/ 0

40

80

120 FRACTION

0 50

100

FIG. 3. Elution patterns showing that Peaks A and B obtained from DEAE-cellulose were equivalent to Peaks A and B, respectively, from DEAE-Sephadex. The proteins eluted from DEAEcellulose in the areas of the two broad bars were pooled separately, concentrated by ultrafiltration, and applied to a DEAE-Sephadex column. A composite of the elution patterns of Peaks A and B, although separately applied and eluted from DEAE-Sephadex, is shown on the right. The dimensions of each column were Enzyme was 150 X 9 mm and fractions of 0.75 ml were collected. eluted from DEAE-cellulose with a linear gradient consisting of 50 ml of the phosphate-Tris buffer3 containing 0.03 M KC1 in the mixing chamber and a reservoir of 50 ml of the same buffer containing 0.12 M KCl. The flow rate was 20 ml per hour. The DEAE-Sephadex system was identical except for the KC1 concentrations which were increased to 0.05 M in the mixing flask and 0.27 M in the reservoir; flow rates of 10 to 15 ml per hour were atof the effluent tained. The arrows represent the conductivities solutions at 2 and 5 mmho for DEAE-cellulose and 3 and 9 mmho for DEAE-Sephadex.

/’

,3

/’,’

,’

1 /,’ ,,’ ,/’ /

025

i j,“’ c /” ” 1 y”

01

A 1

50

s

,’I,’

Li

5 =I v)

100 FRACTION

50

I

)-

/

()

100

NUMBER

FIG. 4. Effect of storage at 4” on the DEAE-Sephadex elution patterns of Peak A (left) and Peak B (right). The top curves represent the samples prior to storage. The lower curves were obtained after 2 days (Peak A) and 10 days (Peak B) at 4”. Details of the chromatography on DEAE-Sephadex are identical with those outlined in Fig. 3. The arrows represent conductivity of the effluent solutions at 3 and 8 mmho for each gradient.

at pH 6.5 containing 50 mM thioglycerol was added to 1.5 mg of enzyme in 100 ~1, prepared by concentration as described. The To this solution, 90 mg of ammonium sulfate were added. resultant suspension was centrifuged at 4” and at approximately 2000 x g in a 0.5-ml centrifuge tube; the supernatant fluid was discarded. The residue was triturated at 0” with 50 ~1 of a solution which was 55oj, saturated with respect to ammonium sulfate, containing 0.1 M potassium phosphate at pH 6.5, 50 mu After 10 min the suspension was thioglycerol, and 25 ‘% glycerol. centrifuged and the supernatant fluid was set aside at room temperature. The residue was treated with a 51% saturated ammonium sulfate solution in an identical fashion and subsequently with 47, 43, 39, and 35% ammonium sulfate solutions. Cloudiness was apparent in the tubes containing the 55% and 51% saturated ammonium sulfate within 12 hours and crystal formation was evident when aliquots of the suspension were viewed with an oil immersion lens and a dark-field condenser. Over a period of several days the crystal size increased. Fig. 2 shows a cluster of large crystals, approximately 10 p in the largest axis, from the 55% ammonium sulfate extract after 7 days at room temperature. The major portion of the crystal population was small and about 1 to 3 p in the longest axis. Repeatedly, significant loss of enzyme activity was observed to result from the crystallization step. For the preparation cited, only 25% of the initial activity was retained 10 days after crystallization. However, the crystalline material had twice the specific activity of its supernatant fluid.

Downloaded from www.jbc.org by guest, on October 17, 2011

.02

;/ It

H

Issue of November

10, 1967

C. R. Xteinman and W. B. Jakoby

RESULTS

Evidencefor Proteolysis-Initial

DISCUSSION

A minimum of three protein species, active in the potassiumactivated and DPN-linked catalysis of aldehyde oxidation, have been isolated from autolysates of bakers’ yeast. Although these three can readily be separated from each other, the relationship among them is not clear. However, it is suggested that proteolysis is one factor in the origin of the multiple forms of aldehyde dehydrogenase. That proteases are present in autolysates of yeast has been documented (7, 8) and similar problems resulting from proteolysis have been noted (cf. 9, 10). The evidence presented here is based on two observations. Primarily, there was a loss in enzyme, particularly during the early stage of the purification procedure, which was curtailed by the addition of the esterase inhibitor, phenylmethylsulfonylfluoride. Secondarily, purification by methods which would tend to decrease the possibilities of extensive hydrolysis, i.e. extraction by sonic oscillation, omission of a heat step, and rapid processing of the enzyme, yield A protein as the only active species. Although the latter experiment could have represented a failure to extract other active species, it appears reasonable to assume that proteolysis is a factor in the loss of enzyme activity and, possibly, in the conversion of A protein to other active species. The finding that A protein was converted to B protein is not easily explained by the information available. An obvious possibility is that of a proteolytic enzyme contaminating A protein, possibly one which is not inhibited by eeterase reagents. Such a proteolytic enzyme would be present in sufficient concentration to cause partial digestion, thereby creating B protein, but insufficient to be recognized as a contaminant by the criteria of ultracentrifugation pattern, disk gel electrophoresis, and chromatography on Bio-Gel P-200. Since storage of protein A for even short periods of time results in the accumulation of protein B, it should be recognized that the hydrodynamic experiments were actually performed on a mixture of both species despite the findings that these studies suggest homogeneity. Acknowledgments-We gratefully acknowledge the guidance and participation of William Carroll and Ellis Mitchell in the hydrodynamic studies. REFERENCES 1. JAKOBY, W.B.,inP.D. BOYER, H. LARDY,AND K. MYRBHCK (Editors), The enzymes, Vol. 7, Ed. 2, Academic Press, New York, 1963, p. 203. 2. BLACK, S., Arch. B&hem. Biophys., 34, 86 (1951). 3. BLACK. S.. in S. P. COLOWICK AND N. 0. KAPLAN (Editors). Methods‘in enzymology, Vol. 1, Academic Press, New York; 1955, p. 508. 4. ORNSTEIN, L., Ann. N. Y. Bad. Sci., 121, 321 (1964). 5. LOWRY,~. H., ROSEBROUGH,N. J., FARR, A.L., ANDRANDALL, R. J., J. Biol. Chem., 193, 265 (1951). 6. WARBURG, O., AND CHRISTIAN, W., Biochem. Z., 310, 384 (1942). 7. LENNEY, J. F., J. Biol. Chem., 221, 919 (1956). 8. JUNI, E., AND HELM, G. A., Bad. Proc., p. 132 (1967). 9. CHIBA, H., SUGIMOTO, E., AND KITO, M., Bull. Agr. Sot. Japan, 24, 558 (1960). 10. SCHULZ, I. T., GAZITH, J., AND COLOWICK, S. P., Fed. 24, 224 (1965).

Chem. Proc.,

Downloaded from www.jbc.org by guest, on October 17, 2011

attempts at purification of the enzyme clearly revealed a continuing loss of activity as a function of time which could be compensated for only partially by the use of a mercaptan and high concentrations of glycerol. Phenylmethylsulfonylfluoride was added during extraction on the assumption that proteolysis was occurring and resulted in a 4to 5-fold increase in recoverable activity. However, the difficulty in using this relatively water-insoluble esterase inhibitor was magnified by adding it to intact yeast at a low temperature with resulting partial precipitation and poor distribution of the inhibitor. Under such conditions, the enzyme obtained after salting out the protein with ammonium sulfate continued to be inactivated. Whereas 98% and 69% of activity were lost after storage of the ammonium sulfate fraction for 7 hours at 22” and O”, respectively, the addition of 50 pg of phenyhnethylsulfonylfluoride per mg of protein resulted in the loss of less than 15% of activity; n-propanol, the diluent, had no effect by itself. One small-scale preparation of enzyme was obtained by subjecting 5 g of nitrogen-treated yeast to sonic oscillation followed by Steps 4 (salt precipitation) and 5B (DEAE-Sephadex) of the purification procedure; a total of 3 hours was required between extraction and charging the DEAE-Sephadex column. Under these conditions, which included the use of esterase inhibitor, only one protein species was obtained, which was identified as Peak A by the characteristic conductance at which elution took place. Peaks A and B-The active proteins from DEAE-Sephadex, labeled Peak A and Peak B, appear to be equivalent to that in Peaks A and B, respectively, obtained by elution from DEAEcellulose (Fig. 3). However, a slow but definite conversion of Peak A to Peak B occurs. In the experiment detailed in Fig. 4 it is evident that the A protein was converted, in part, into catalytically active B protein during the 2-day period required to concentrate and rechromatograph the material. However, the B protein was stable to such a procedure which includes storage at 4” for 10 days. In both cases there was little, if any, loss of activity during the course of the entire procedure since more than 90% of the activity applied to each column was recovered in the eluates. Preparations of the B protein had half of the activity of A protein. Molecular Weight-Sedimentation and diffusion studies of solutions of the enzyme, i.e. Peak A, were carried out in 0.05 M potassium phosphate at pH 7.0 containing 25% (weight per volume) glycerol and 0.1 M thioglycerol; by direct determination this medium had a viscosity, relative to water at 25”, of 2.508 and a density of 1.0784. The sedimentation constant was independent of protein concentration over the range tested. With protein concentrations of 1.7 mg, 2.2 mg, and 5.0 mg per ml, szu,25values of 9.64 S, 9.67 S, and 9.64 S, respectively, were obtained. In each instance, a single, symmetrical peak was observed. The diffusion coefficient, corrected to water at 25”, was 4.407 x 10-r cm2 see-l for a protein concentration of 6 mg per ml. Based on the diffusion coefficient, an s&,,~ of 9.65 S, and an assumed partial specific volume of 0.73 ml per g, the molecular weight was calculated to be 200,000.

5023