THE .JOURN~L OF BIOLOGICAL CHEYISTRY Vol. 240, No. 6, June 1965

Printed in U.S.A.

Purification, Composition, and Molecular Weight of the

p-G1 t d a ac osi ase of Escherichia coli K12

GARY R. CRAVEN, EDWARD STEERS, JR., AND CHRISTIAN B. ANFIKSEN

From the Laboratory of Chemical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 2001/,

(Received for publication, December 28, 1964)

The currently accepted concepts regarding the nature of the genetic regulation of protein synthesis are based, to a large extent, on genetic studies of the “lac operon” of Escherichia coli K12 (1). These, and other investigations on similar systems such as the histidine operon of Salmonella (2), have suggested the opera- tion, at the genetic level, of a complex process of repression, derepression, and sequential synthesis of metabolically linked enzymes.

Since genetic experimentation can only suggest but not prove the consequences of mutation, we have initiated studies on the chemical nature of ,&galactosidase produced by the K12 strain of E. coli and a number of its mutant forms. Previous work on the chemistry of the P-galactosidase of E. coli has been carried out principally by Wallenfels et al. (3-5). Their studies have been confined, however, to the ML30 strain of E. coli which is not suitable for genetic experimentation. Such problems as the location of the “operator” locus with relation to the structural gene cont,rolling the sequence of the enzyme, the consequences of “polarity” mutations, and the chemical nature of intracis- tronic complementation can, we feel, only be approached through a relatively detailed study of the covalent structure of the final product of genet,ic regulation, the enzyme itself.

The purification of P-galactosidase from lysates of E. coli was first studied in a systematic way by Cohn (6) and by Kuby and Lardy (7). Later studies by Wallenfels et al. (4) and by Hu, Wolfe, and Reithel (8) have led t’o the isolation of material of relatively high purity. However, as discussed in t,he present paper, certain minor contaminants of lower molecular weight and somewhat higher lysine content than ,&galact,osidase could only be removed by the application of one of several additional purification steps. The final material described here does not show detectable contamination with minor components upon ultracentrifugal and electrophoretic analysis.

This communication summarizes the purification, criteria for homogeneity, and some of the physical characteristics of the p-galactosidase of E. coli K12. The accompanying paper (9) is concerned with more detailed studies on the chemical architec- ture of the enzyme.

EXPERIMENTAL PROCEDURE

Materials

Chemicals used for polyacrylamide gel electrophoresis were obtained from Canal Industrial Corporation, Bethesda, Mary- land. The iodoacetic acid used for carboxymethylation was

obtained from Eastman Organic Chemical Company and was routinely recrystallized from ether prior to use. o-Nitrophenyl- fi-n-galactopyranoside was purchased from the California Cor- poration for Biochemical Research. Standard solutions of amino acids used for the automatic amino acid analyses were from the Beckman-Spinco Corporation. 2-Mercaptoethanol was obtained from Eastman Organic Chemical Company.

DEAE-cellulose was obtained from California Corporation for Biochemical Research and both Sephadex G-200 and DEAE- Sephadex A-50 were obtained from Pharmacia, Uppsala, Sweden. These materials were washed with 1 N NaOH and 3 N HCl, freed of ‘(fines” by allowing them to settle several times after suspen- sion in water, and thoroughly equilibrated wit’h the proper buffer before columns were prepared.

Methods

All polyacrylamide gel electrophoresis experiments reported here were performed at 4-6” with 7.5% standard gel as described in the manual supplied by the Canal Industrial Corporation. Normally, 16 gel tubes (8 x 0.6 cm, inner diameter) were run at one time with a constant current of 3 ma per tube.

Sucrose density cent.rifugation (10, 11) was carried out with a gradient that covered the range of sucrose concentrations from 20 to 5%. Sucrose solutions were made up in standard buffer (see below) and 2mercaptoethanol was added to a concentration of 0.1 M just prior to t,he preparation of gradients. Centrifuga- tion was carried out for 24 hours at 22,500 rpm in a Spinco, model L, ultracentrifuge with a SW rotor. Approximately 1 ml of protein sample containing from 15 to 30 mg of protein was layered over the sucrose solution.

Strains and Growth of Bacteria

The experiments reported here have all been performed with P-galactosidase isolated from E. coli K12 strain 3300. This strain, which is constitutive for galactosidase, was obt,ained from Dr. J. Monod and Dr. F. Jacob. The strain can grow on minimal salts medium with added vitamin 131. For growth of large cultures the following medium (per liter) was used: KH2P04, 3 g; Na2HP04, 6 g; (NH&Sod, 2 g; NaCl, 3 g; MgSO+ 0.05 g; FeS04, 5 mg; glycerol, 20 ml; vitamin RI, 0.5 mg; and Difco casamino acids, 3 g. The pH was adjusted to 7.2. Glycerol, vitamin B1, and MgS04 were autoclaved separately.

The bacteria were normally grown in 300.liter lots. Under t,hese conditions it was found that significant yields of cells could only be achieved when casamino acids (Difco) were added to the

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June 1965 G. R. Craven, E. Steers, Jr., and C. B. Anfcnsen

medium. The 300.liter fermenter available at the National Institutes of Health was innoculated aseptically with a l-liter log phase culture of bact,etia. Both the initial starting cultures and the final 300.liter culture were routinely screened for con- taminating organisms. Growth was continued for 16 hours at 37”.

The bacteria were harvested in a Sharples centrifuge. The average yield of wet weight cells was 2500 to 3000 g/300 liters.

Buffers

The standard buffer used for preparing solutions of enzyme preparations throughout this work contained 0.01 M Tris-acetate and 0.01 M MgC12, pH 7.5 (Buffer A). When crude enzyme was being handled in a routine fashion, 0.01 M 2-mercaptoethanol and 0.01 &r NaCl were present. How-ever, when enzyme of highest purity was being studied, the 2-mercaptoethanol and NaCl con- centrations were raised to 0.1 111 (Buffer B).

Carboxymethylation

The carboxymethylation of galactosidase was carried out as previously described (12) with the modification that 8 M urea was used not only for reduction of the protein with mercapto- ethanol, but also during the alkylation reaction. The protein was dialyzed against 0.1 M NH4HC03 and then made 8 M in urea by the addition of solid, recryst’allized urea. 2-Mercaptoethanol was t,hen added to a level of 0.1 M and the solution was incubated at 45” for 3 to 4 hours. After the incubation, iodoacetate was introduced in a 5-fold molar excess over the mercaptoethanol. The reaction mixture was kept at pH 8.2 by the constant addi- tion of 6 N NaOH and was allowed to proceed for 15 minutes, before termination by the addition of excess mercaptoethanol. The final product was dialyzed. The carboxymethylat,ed galac- tosidase occasionally precipitated upon dialysis, in which event the total suspension was again made to 8 M in urea and re- dialyzed against ammonium bicarbonate solut’ion.

Enzyme Assay and Definition of Units

The fi-galact’osidase was assayed for enzyme activity with the synthetic substrate, o-nitrophenyl-fl-n-galactopyranoside. For routine work, the buffer outlined above was used. However, when a standard evaluation of the specific activity of an enzyme preparation was desired, the conditions were altered to coincide with those employed by Cohn (6) for enzyme assay. One unit is defined as that amount of enzyme which hydrolyzes 10-Q moles of o-nitrophenyl-/?-n-galactopyranoside in 1 minut’e at 28” in 0.1 M

sodium phosphate buffer (pH 7.0), containing 10e3 M Mg++ and 0.1 M 2-mercaptoethanol. The concentration of o-nitrophenyl- P-n-galactopyranoside solution was 0.70 g per liter of buffer. Mercaptoethanol was added immediately prior to assay.

Acid Hydrolysis and Amino Acid Analysis

The lyophilized protein was suspended in constant boiling, redistilled hydrochloric acid (approximately 5.7 N) in a hydroly- sis tube. The tube was constricted and thoroughly evacuated for 15 minutes. After evacuation t,he tube was sealed and placed at 100” for the desired time (routine analysis was for 20 hours). The hydrolysate was t,aken rapidly to dryness in a vacuum and amino acid analysis performed by the method of Spackman, Moore, and Stein (13), wit,h t,he Spinco automatic amino acid analyzer.

Immunodi$usiow-The antisera used in this and the following paper were prepared as outlined by Balbinder and Preer (14). Two preparations of P-galactosidase were employed as antigens for immunization, one purified by chromatography on DEAE- Sephadex and the second on DE*iE-cellulose. The agar gel double diffusion technique of Preer (15, 16) was used for all immunological assays. In this method, three layers are formed (approximately 4 mm each) in 2-mm (inner diameter) Pyrex tubes. The lower layer is antiserum, the upper layer antigen, and the middle layer agar. The reactants diffuse into the agar forming a precipitin zone in the region of their equivalence. The developed tubes are visually analyzed with the aid of a stereo- scopic binocular microscope (0. 7X t’o 3X, zoom ocular) with a 10X objective.

Electrophoresis-The electrophoretic behavior of the galac- tosidase prepared by chromatography in DEAE-Sephadex was studied in a Perkin-Elmer model 238 Tiselius apparatus. Buffer B was employed for all electrophoretic runs, at 0.1 M

ionic strength. The temperature was maintained at O-1”. The sample to be analyzed was dialyzed against 2-liter volumes of buffer for 48 to 76 hours wit’h 3 buffer changes.

Ultracentrifugal Analysis-The Spinco model E analytical ultracentrifuge, equipped with the RTIC temperature control unit and Raleigh optical systems, was employed. All analyses were carried out in Buffer B. Velocity centrifugation was con- ducted with the st,andard single sector cell at 59,780 rpm. All calculations were corrected to water at 20” and zero protein concentration according to the procedure described by Schach- man (17). The molecular weight was determined by two of the equilibrium techniques currently available. The first, or low speed equilibrium technique, was employed as described by Klainer and Kegeles (18). The second, or high speed equilibrium technique, has recently been described by Yphantis (19). All ultracentrifugal analyses were carried out at 20”. The low speed equilibrium runs (at 2,531 rpm) and high speed Yphantis analyses (7,447 rpm and 8,227 rpm) were carried out utilizing the Spinco An-J rotor. The standard double-sector interference cell, as well as the new six-channeled special cell (Spinco Corpora- tion) designed by Yphantis were used for the equilibrium studies.

Preparative Electrophoresis -Preparative electrophoresis was performed in the Brinkmann continuous flow electrophoretic separator (Brinkmann Instrument Company, Great Neck, Long Island, New York). The samples t’o be applied were dissolved in the same buffer as that employed for making the buffer curtain; 0.01 M Tris, 0.01 &f NaCl, 0.01 $1 MgCl2, and 0.01 M X-mercapto- ethanol, pH 7.5. The applied volt’age was 1500 volts and the current was 200 ma.

RESULTS

Puri$cation of P-Galactosidase

In out,line, the procedure described below involves ammonium sulfate precipitation of the crude extract followed by Sephadex G-200 filtration. The active fraction from the gel column is applied to a column of DEAE-Sephadex A-50 and eluted with a NaCl gradient as described by Hu, Wolfe, and Reithel (8) for DEAE-cellulose columns. The major, central portion of the active eluted peak is free of detectable contamination upon examination by polyacrylamide gel electrophoresis, free boundary

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2470 Puri$cation and Properties of p-Galactosidase Vol. 240, No. 6

“0.9 I.1 1.3 1.5 I7 I.9 2.1 2.3 2.5 2.7 -

VOLUME ILilers~

FIG. 1. Gel filtration on Sephadex G-200 of the 40yo ammonium sulfate-precipitated fraction (approximately 4 g of protein in 200 ml of Buffer A). The column dimensions were 10 X 40 cm. The column bed contained 157, cellulose powder (ZOO to 400 mesh) and was eauilibrated with Buffer A (see “Exoerimental Proce- dure”). Gravity flow was used, taking care- to keep the hy- drostatic pressure on the column bed as low as possible. Frac- tions of 10 ml each were collected. - - - optical density at 280 mp; --, units of p-galactosidase activity:

electrophoresis, high speed equilibrium measurements in the ultracentrifuge, and immunological study.

Other purification procedures have also been applied in the last stages of purification such as preparative sucrose density centrifugation and electrophoretic separation in the Brinkmann apparatus. Similarly homogeneous material can be obtained although in smaller amounts and with less convenience. The use of DEAE-Sephadex columns appears to us to be the method of choice.

Purijication

Preparation of Crude Extract of B. coli-In a routine purifica- tion of fl-galactosidase, approximately 1 Kg (wet weight) of un- washed, frozen E. coli cells mere suspended in 2 liters of the standard buffer (see “Experimental Procedure”). The sus- pended cells were passed twice through a continuous flow cell on the Branson sonifier (Branson Instruments, Inc., Stamford, Connecticut)’ which was kept at 0”. The pH of the resultant slurry was usually 5.5 to 6.0. Readjustment of the pH to 7.5 prior to the second sonication did not appear to improve the yield of enzyme and was omitted. The disrupted cell suspension was centrifuged overnight in an International refrigerated cen- trifuge at 2500 X g, and the supernatant solution was used directly in the subsequent purification.

Ammonium Sulfate Precipitation

The crude extract was adjusted to pH 7.5 and the P-galactosi- dasc activity precipitated by the addition of solid ammonium sulfate to 40% saturation at room temperature. The increase in specific activity accomplished by this step was approximately a-fold.

1 In large scale preparations of p-galactosidase employing be- tween 3 and 25 Kg-of -frozen cells, cell rupture has been achieved in a Laboratorv Sub Micron Disnerser. Manton-Gaulin Manu- facturing Company, Everett, Massachusetts.

Throughout the purification, ammonium sulfate precipitation was utilized for concentration of the enzyme from solution. All enzyme solutions were stored under ammonium sulfate.

Filtration through Sephadex G-ZOO

The 40% ammonium sulfate precipitate was collected by cen- trifugation and dissolved in less than 150 ml of the standard buffer.2 The resulting solution was dialyzed overnight against the standard buffer and the small amount of insoluble material that separated was removed by centrifugation. The centrifuged solution was then layered on a column of Sephadex G-200 (10 x 40 cm) containing 15 to 20% by weight of cellulose powder and previously equilibrated with the standard buffer. The added cellulose did not affect the general characteristics of the filtration pattern but greatly increased the flow rate. Fig. 1 illustrates a typical elution pattern. The main peak of activity emerges from the column shortly after the front, which is characterized by considerable turbidity. It would be expected that a protein as large as P-galactosidase would filter with the front on G-ZOO columns. The significant retention noted here suggests adsorp tion, an effect also noted in the absence of added cellulose powder. The fraction of the active material eluted with the turbid, front- running material remains associated with this material upon refiltration and may represent either a highly polymerized form of the protein or a fraction tightly bound to the nucleic acid components of the mixture.

The gel filtration step afforded a major purification as is illus- trated by the polyacrylamide gel electrophoresis patterns in Fig. 2 and generally resulted in an increase in specific activity of 3- to 4-fold over the ammonium sulfate precipitate. The total protein in the pooled tubes was precipitated by the addition of ammonium sulfate to 40% and the precipitate dialyzed against standard buffer for the following purification step. The pooled fractions from G-200 Sephadex columns generally accounted for approximately 80% of the activit’y units originally applied and normally showed a specific activit’y of 1.8 to 2.0 X lo5 units per mg.

DEAE-Sephadex Chromatography

A column of DEAE-Sephadex 12-50, medium (4 X 25 cm), was equilibrated with standard buffer containing 0.01 ~1 mer- captoethanol. The dialyzed material from the Sephadex G-200 column was adsorbed to the column and eluted as summarized in Fig. 3. The specific activity of the enzyme prepared in this manner was on the order of 340,000 units per mg, considerably higher than that obtained by any other technique. DEAE- cellulose chromatography, under t’he same elution conditions, routinely has given material of approximately 250,000 units per mg of protein. Furthermore, while polyacrylamide gel elec- trophoresis showed considerable contamination in the latter preparations, the DEAE-Sephadex product showed only the galactosidase band.

Gel electrophoresis patterns of samples taken along the active peak are shown in Fig. 4. These gel patterns demonstrate the presence of minor impurities in some leading and trailing tubes. The central portion of the active peak, which appeared homo-

2 The ammonium sulfate precipitate from more than 1 Kg of cells may be applied to G-200 columns of the dimensions described here so long as the density of the final dialyzed protein solution is not high enough to cause inversion on the column.

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June 1965 G. R. Craven, E. Steers, Jr., and C. B. Anjinsen

FIG. 2. Purity at various stages in the purification of p-galactosidase as monitored by polyacrylamide gel electrophoresis. The total number of fl-galactosidase units applied to each gel was increased with each stage in the purification so that impurities could be more readily seen. The gels used here are standard gels (running at pH 8.5), the negative pole being at the end occupied by the spacer and sample gels (neither are shown in the photographs).

geneous on disk gel electrophoresis (Fig. 4), was collected, con- centrated by vacuum dialysiq3 and examined in the ultra- centrifuge (Fig. 5). This material was also examined in the Perkin-Elmer moving boundary electrophoresis apparatus at a concentration of 20 mg of protein per ml. Even at this high concentration only traces of electrophoretic impurities (a small trailing shoulder) could be seen (Fig. 6).

Further evidence of homogeneity was obtained through the use of antiserum prepared against a galactosidase preparation of a lower degree of purity (from a DEAE-cellulose column). This antiserum showed, in addition to a strong precipitin band in Ouchterlony and Preer double diffusion tests (corresponding to galactosidase), five to seven other antigenic components when analyzed against the homologous galactosidase preparation. However, when this antiserum was used in double diffusion experiments against the final product of DEAE-Sephadex chro- matography, only a single antigenic component was observed over a wide range of concentrations.

The yield of galactosidase of high purity from DEAE-Sephadex columns was usually about 50y0 of the activity applied. How- ever, there appeared to be virtually no loss in the total units of activity emerging from the column (in contrast to DEAE-cel-

lulose, where we have normally observed a 15 to 20% loss in total enzyme units).

3 The sample was placed in dialysis tubing open to the atmos- phere and dialyzed against buffer which was under reduced pres- sure.

IC

FRACTION NUMBER

FIG. 3. Fractionation of &galactosidase on diethylaminoethyl Sephadex A-50. The column dimensions were 3 X 25 cm, and approximately 750 mg of p-galactosidase (dialyzed against Buffer A) were adsorbed to the column which had been previously equili- brated with Euffer A. Fractions of 7 ml each were collected. A sodium chloride gradient was generated with a 500-ml mixing compartment containing Buffer A, into which Buffer A containing 2% added sodium chloride was allowed to drip by gravity. - - -, optical density at 280 rnp; -, units of p-galactosidase activity.

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2472 Purification and Properties of p-Galactosidase Vol. 240, No. 6

FIG. 4. Homogeniety as determined by polyacrylamide gel electrophoresis, of various fractions from the DEAE-Sephadex column, as indicated in Fig. 3. The numbers given below the photographs represent tube numbers shown in Fig. 3. Exactly 25 ~1 from each tube was used for the polyacrylamide gel electrophoresis. The conditions of electrophoresis are given in Fig. 2 and under “Experimental Procedure.”

r

FIG. 6. Moving boundary electrophoresis of ,&galactosidase purified on DEAE-Sephadex. Protein concentration was 20 mg per ml, in Buffer B (see “Experimental Procedure”). The tracing of the original picture shown here was taken after 6$ hours at a constant current of 10 ma. The electrophoresis was carried out in a Perkin-Elmer model 238 electrophoresis apparatus.

-Ascending

Alternative Pur$cation Procedures

Before the superiority of DEAE-Sephadex chromatography had been discovered, several alternative methods wereused to improve the purity of the material obtained by chromatography on DEAE-cellulose. Each of these procedures has been employed

FIG. 5. Schlieren pattern, obtained in the Spinco model E to prepare the materials used in some of the studies to be de- ultracentrifuge, of p-galactosidase after purification on DEAE-

The picture was taken at 24 minutes after reaching a scribed below and in the accompanying paper (9), since the

Sephadex. speed of 59,780. The enzyme had been previously dialyzed against

degree of contamination is small in all cases.

Buffer B (see “Experimental Procedure”). Enzyme concentra- The degree of purity obtained with DEAE-cellulose was

tion was 7.2 mg per ml. usually on the order of 80 to 85% as judged by ultracentrifugal

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June 196.5 G. R. Craven, E. Steer-s, Jr., and C. B. Anjinsen 2473

TABLE I Amino acid composition of p-galactosidase

Amino acid

Lysine ............ Histidine ......... Arginine ......... S-carboxymethyl-

cysteine ......... Aspartic acid. ... Threonine ........ Serine ............ Glutamic acid ..... Proline ........... Glycine. .......... Alanine. .......... Valine ............ Methionine ...... Isoleucine. ........ Leucine. ........ Tyrosine. ......... Phenglalanine. .... Tryptophand ......

- 16 hr

0.087 0.0856 0.091 0.089 0.096 0.099* 0.092 0.091* 0.108 0.111 0.112 0.113 0.118 0.127 0.122 0.109 0.232 0.234 0.228 0.224 0.232 0.230 0.232 0.232

pmoles pmoles 70

0.098 2.48 0.114 3.11 0.230 6.30

0.056 0.058 0.065c 0.06~5~ 0.058 0.062 0.05? 0.059 0.060 1.64 0.390 0.387 0.383 0.403 0.387 0.384 0.385 0.362 0.385 10.50 0.198 0.197 0.191 0.196 0.189 0.189 0.182 0.182 0.202 5.52 0.201 0.199 0.193 0.196 0.177 0.179 0.162 0.166 0.208 5.69 0.440 0.440 0.441 0.446 0.444 0.440 0.449 0.440 0.443 12.11 0.205 0.212 0.204 0.220 0.203 0.214 0.212 0.206 0.209 5.70 0.266 0.263 0.265 0.264 0.263 0.266 0.265 0.265 0.264 7.22 0.289 0.289 0.286 0.304 0.290 0.290 0.291 0.291 0.291 7.96 0.214 0.213 0.224 0.231 0.232 0.233 0.232 0.232 0.232 6.35 0.081 0.076 0.059 0.068 0.071 0.071 0.077 0.085 0.076 2.08 0.131 0.131 0.140 0.142 0.145 0.147 0.147 0.157 0.149 4.07 0.341 0.340 0.336 0.348 0.337 0.351 0.343 0.346 0.344 9.40 0.114 0.111 0.110 0.119 0.111 0.116 0.111 0.110 0.113 3.09 0.136 0.136 0.142 0.141 0.138 0.144 0.136 0.138 0.139 3.80

Sample

32 hr 56 hr

a Calculated on the basis of an average residue molecular weight of 114.9. . . . h These analyses were corrected for physical loss by normalization to glutamlc acid. . . c These values were not included in the average since the S-carboxymethylcysteme position on the chromatogram of these analyses

overlapped a small component which normally falls between S-carboxymethylcysteine and aspartic acid. d Approximately 35 residues/135,000 g (see Table II).

80 hr

Average

-

( re

-

:alculated :sidues per 135,oooa

29 36 74

19 123

65 67

142 G7 85 93 75 24 48

110 36 45 35

analysis. It was found that this could be raised to 95c/, or .25 I I I I greater either by sucrose density gradient centrifugation or by i curtain electrophoresis in the Brinkmann continuous flow elec- g _ trophoretic separator. Contaminants demonstrable by ultra- - centrifugation in the DEAE-cellulose column product were essentially absent after either of these procedures. A third i - approach to purification involved the property of carboxy- : methylated galactosidase, in which all sulfhydryl groups had $ _ been reacted with iodoacetic acid, of precipitation after dialysis against distilled water from a 5 M guanidinium chloride solution. 0 20 40 60 80 100

During such dialysis, contaminating components showing un- HYDROLYSIS TIME (Hours)

usually high absorption at 260 rnp were left in solution. The FIG. 7. Destruction of serine and threonine during hydrolysis

resulting insoluble product gave an amino acid analysis that was of carboxymethylated p-galactosidase. Hydrolysis was per-

similar to purified galactosidase samples prepared by other formed in thoroughly evacuated sealed tubes containing approxi- mately 0.5 mg of protein in I ml of constant boiling HCl.

methods and showed only a single electrophoretic peak upon frer boundary electrophoresis. plained loss of methionine and was therefore not included in the

average for this residue.

Amino Acid Analysis The corrected values for serine and threonine were calculated by extrapolation to zero time as shown in Fig. 7. It should be

i\ solution of carboxymethylated P-galactosidase, which had noted that the losses of threonine and serine during 80 hours of been prepared by DEAE-cellulose chromatography and subse- hydrolysis (relative to the extrapolated values) were only 6.5% quent sucrose density gradient centrifugation, was divided into and 15%, respectively. This unusually small loss may be due to eight equal aliquots and placed in hydrolysis tubes. The sam- the apparently complete absence of carbohydrate in the protein

ples were lyophilized and dissolved in constant boiling HCl preparation.3 Carbohydrate analyses were performed by the (5.7 N). The tubes were then evacuated, sealed, and heated at phenol-sulfuric acid method of Dubois et al. (20). Wallenfels 110” for various lengths of time. The results of amino acid and Malhotra (3) have also reported the absence of carbohydrate

analysis of these eight samples are summarized in Table I. The in preparations of P-galactosidase from E. coli of the ML-309 micromolar yields of the amino acids have been averaged in strain. Column 10 with the exception of the results for valine, leucine, The absence of carbohydrate may also account for the rela- and isoleucine for which only the 56 and 80-hour results were in- tively high recovery of tryptophan after acid hydrolysis.4 Al- eluded in the averages. The 32-hour sample showed an unex- 4 Prof. A. Neuberger, personal communication.

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2474 Purification and Properties of P-Galactosidase Vol. 240, Ko. 6

TABLE II sidase used for amino acid analysis, and assuming 100% recovery Summary of analyses for tryptophan in P-galactosidase of residues on the amino acid analyzer, the extinction coefficient,

at 280 mu for the nure enzyme may be calculated to be 2.09 mg Method of analysis

Acid hydrolysis and amino acid analysisa. Alkaline hydrolysis and amino acid analy-

sisa.................................... Spectral analysis in 0.1 M NaOH (21) a. N-Bromosuccinimide titration (22) b

Tryptophan content, residues per 540,000 g

86

114 140 157

L, per ml employing a l-cm light path. The ratio of the absorbance at 280 rnp and 260 rnp is 1.96 which compares well with the value of 1.92 reported by Wallenfels and Malhotra (3). It was ob- served that the increased purity of the enzyme preparation at each step in purification, as determined by ultracentrifugation and electrophoresis, was reflected in an increase of this ratio.

y Carboxymethylated galactosidase was used in these analyses. Content of Sulfhydryl Groups and D&&de Bonds

* Analysis kindly performed by Dr. Michael Green on native The amino acid analyses of P-galactosidase presented above galactosidase. show the presence of approximately 76 residues of half-cystine

TABLE III

Reactivity to iodoacetic acid of sulfhydryl groups of @-galactosidase in presence and absence of dissociating solvents

Conditions of alkylation

Amino acid Without reduction After reduction in 8 P urea

7 M guanidine

rmole wde % Lysine .................................. 0.161 3.1 Histidine ... ............................ 0.176 3.4 Arginine ................................. 0.351 6.7 S-Carboxymethylcysteine. ............... 0.081 1.6 Aspartic acid. .......................... 0.571 11.0 Threonine” .............................. 0.280 5.4 Serine” .................................. 0.385 7.4 Glutamic acid, .......................... 0.656 12.6 Proline ................................. 0.305 5.9 Glycine ................................. 0.436 8.4 Alanine. ................................ 0.415 8.0 Half-cystine ............................ Absent Valine ................................... 0.279 5.3 Methionine ......... .................... 0.101 1.9 Isoleucine ............................... 0.158 3.0 Leucine ................................ 0.466 8.9 Tyrosine. ............................... 0.148 2.8 Phenylalanine ........................... 0.177 3.4

a Uncorrected for destruction during acid hydrolysis.

ternative methods for estimation of tryptophan did reveal, however, that a certain amount of destruction of this amino acid had occurred as shown in Table II.

Since the estimation of tryptophan in proteins is generally unreliable, short of complete sequence determination, the present results can only be taken to indicate a likely range of tryptophan content. Spectral analysis in alkali (21) and N-bromosuc- cinimide titration (22), monitored by spectral analysis, are probably the most dependable of the methods listed and a value of 140 to 160 residues per 540,000 is therefore the best present estimate.

Amino acid analyses have also been performed on performic

acid-oxidized P-galactosidase. These analyses indicated the presence of 73 residues of half-cystine (determined as cysteic acid) in 540,000 g of protein, a value which compares favorably with the S-carboxymethylcysteine determinations (76 residues/ 540,000 g as shown in Table Is).

On the basis of the absorbance of stock solutions of P-galacto-

5 The residue calculation in Table I assumes a mole molecular weight of 135,000 (quarter molecule) as discussed in Reference 9.

10 M ureaa Exwriment 1 Experiment 2

&mole mole %

0.399 7.0 0.089 1.6 0.630 11.1 0.312 5.5 0.400 7.1 0.719 12.7 0.326 5.8 0.469 8.3 0.464 8.2

Trace 0.312 5.5 0.127 0.0 0.102 1.8 0.040 1.9 0.179 3.2 0.075 3.6 0.520 9.2 0.199 9.4 0.160 2.8 0.064 3.0 0.200 3.5 0.064 3.0

pm&

0.060 0.068 0.139 0.033 0.238 0.118 0.126 0.265 0.125 0.167 0.181

rntole 70

2.9 3.2 G.6 1.6

11.3 5.6 6.0

12.6 5.9 7.9 8.G I

Absent

/LWtlJle rmole %

0.050 2.3 0.065 3.0 0.135 6.2 0.035 1.6 0.248 11.4 0.123 5.7 0.132 6.1 0.025 12.7 0.136 6.2 0.175 8.1 0.185 1 8.G

Absent 0.132 6.1 0.042 1.9 0.075 3.5 0.204 9.4 0.067 3.1 0.083 3.9

per molecule of the protein. It is important to determine the number of these residues, if any, that take part in disulfide linkages. As a first step, samples of galactosidase were alkylated with excess iodoacetate in 7 M guanidine and in 10 M urea without. prior exposure to conditions of reduction.6 No mercaptoethanol was present during the alkylation except the very small amount introduced from the native enzyme solution (final concentration of 2-mercaptoethanol, less than 1O-5 M). As controls, samples of galactosidase solution were alkylated under the same condi- tions except that the enzyme was exposed to prior incubation in 8 M urea with added mercaptoethanol (final concentration. 0.1 M). The alkylations were terminated, after 15 minutes, by the addition of excess mercaptoethanol, the solutions were then dialyzed, and the resulting protein was hydrolyzed for amino acid analysis. Data pertaining to the number of sulfhydryl groups,

estimated as S-carboxymethylcysteine, are summarized in Table III.

6 Alkylation without reduction in 8 M urea yielded only SO”l, as much S-carboxymethylcysteine as in 10 M urea (see Table III).

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.June 1965 G. R. Craven, E. Steers, Jr., and C. B. Anjnsen 2475

These data suggest that most or all of the half-cystine residues in galactosidase are in the free sulfhydryl form and that, within the precision of the analyses, virtually none take part in disulfide bridge formation.

It was also of interest to ascertain the number of free sulfhydryl groups available to alkylation in the absence of denaturing sol- vent such as aqueous urea or guanidine. X series of alkylations was therefore performed on native galactosidase at various con- centrations of urea. Each urea solution was incubated at room temperature for 30 minutes, and aliquots were taken for the assay of residual activity. The solutions were then treated with excess iodoacetic acid for 15 minutes (12) and again assayed for activity. Excess mercaptoethanol was then added to terminate the alkylation reaction. The final product was dialyzed and prepared for complete amino acid analysis, the results of which are presented in Table IV together with the activities of the enzyme at the various stages of reaction. In the experiments summarized in Table IV, no carboxymethylation of lysine or histidine was evident from the amino acid analysis.

Since cysteine is a minor component of galactosidase, the precision involved in the analysis of S-carboxymethylcysteine and of half-cystine is such that the figures given in Table IV represent only approximations. However, it can be concluded that, in galactosidase, a little over half of the total number of cysteine residues are exposed in the native molecule (i.e. acces- sible to iodoacetic acid) and that these sulfhydryl groups are not essential for activity.

Molecular Weight of Native &Galactosidase

Sund and Weber (23) have recently reported a molecular weight for the native enzyme of 518,000, a value considerably less than that determined by Hu, Wolfe, and Reithel (8) and by Cohn (6) (750,000). The major point of discrepancy between the determinations appears to reside in the values for the diffusion coefficient employed in the calculations. We have estimated the molecular weight of pure preparations by sedimentation equilibrium methods, thus avoiding the necessity for measure- ments of diffusion coefficients. The data summarized in Table V include results obtained by low speed equilibrium measure- ments (18) and the high speed equilibrium method of Yphantis

TABLE IV

Effect of carboxymethylation of suljhydryl groups at various con- centrations of urea on enzymic activity of P-galactosidase

Urea concentration

Activity after S-Calboxy-

methylcysteine Half-cystineb

30 min Carboxy- contenta methylation

M % residues/mole

None 100 100 43 2G 1 100 100 43 26 3 35 15 56 21 5 None None 76 Trace 7 None None 76 None

a Assuming 76 residues of half-cystine/540,000 g of enzyme (see Table I).

b Estimated as cystine on the amino acid analyzer. The com- plete analyses, such as were presented in Table I, are not given here in the interest of limiting space. The precision of estima- tion of cystine and S-carboxymethylcysteine was on the order of 10%.

TABLE V

Ultracentrifugal measurements of molecular weight of native p-galactosidase

Experiment Apparent molecular weight

High speed equilibrium (19)a 1 2 3 4 5

Low speed equilibrium (18) 6 7

549,000 525,000 543,000 551) 000 522,000

445,000 b 534,OOOh

n The average apparent molecular weight of high speed equi- librium is 538,000.

b Not corrected for protein concentration.

3.40 , , , / / , , , , , , / , / , , , , ,

3.20 - A

.

:: L 2.60

46.5 4 7.0 47.5 48.0 46.5 x2 (CM2)

FIG. 8. Molecular weight determination of native P-galactosi- dase with the high speed equilibrium method according to Yphan- tis (19). Protein concentration was 0.3 mg per ml in Buffer B. Speed for the run was 7,447 rpm and the temperature was 20.0”. The molecular weight calculated for this run was 521,000.

(19). The results of a typical experiment with the latter method are shown in detail in Fig. 8. The molecular weight values ob- tained in all the experiments shown are in good agreement with that reported by Sund and Weber (23). A value of 540,000 g per mole has been assumed in the calculations presented in this and the accompanying communication (9). Since approximately the same molecular weight was indicated by the low speed equilibrium technique (enzyme concentration, 6 mg per ml) and by the Yphantis method (enzyme concentration, 0.2 to 0.5 mg per ml), the data suggest that galactosidase does not undergo a concentration-dependent dissociation such as that found with glutamic acid dehydrogenase (24). This has been further cor- roborated by sucrose density gradient experiments on enzyme solutions of only IO-fold the concentration normally used for the enzyme assay (0.1 to 0.5 pg per ml). Under these conditions the sedimentation coefficient was estimated to be 168.

DISCUSSION

Two general methods for the purification of /!I-galactosidase have been reported. Wallenfels et al. (4) have modified and extended the procedure of Kuby and Lardy (7), which involves

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2476 Puri@ation and Properties of p-Galactosidase Vol. 240, r\‘o. 6

ammonium sulfate and alcohol precipitation followed by ex- absence. (With mercaptoethanol, only 50% loss in activity tensive recrystallization, and obtained material which appeared is observed after 40 minutes at 55” whereas without mercapto- to be homogeneous in the ultracentrifuge. Hu, Wolfe, and ethanol all activity is lost after 10 minutes at this temperature.) Reithel (8) have applied DEAE-cellulose chromatography, after When our preparation is dialyzed against buffer not containing ammonium sulfate fractionation, to obtain enzyme of high purity. mercaptoethanol, considerable losses in activity occur, even when However, upon examination of the homogeneity of their prepara- the protein is assayed in the presence of the reducing compound. tion by several methods, they demonstrated the presence of as It seems likely, therefore, that the inconsistencies described above much as 15 to 20% contamination which could largely be re- may be the result of differences in the state of oxidation of sulf- moved by preparative electrophoresis. hydryl groups.

The purification procedure described in this communication permits the preparation of gram quantities of /I-galactosidase in relatively few steps and yields a final product which is virtually homogeneous as judged by several criteria. A significant im- provement over previously described methods results from the use of DEAE-Sephadex chromatography. This ion exchange agent allows good resolution of protein components, which on DEAE-cellulose elute together with /%galactosidase. A critical factor in the purification procedure is the use of polyacrylamide gel electrophoresis to monitor the purity of samples taken across the DEAE-Sephadex chromatography elution profile. This technique can reveal the presence of impurities which chro- matograph within, but not coincident with, the fl-galactosidase peak.

SUMMARY

The purified enzyme exhibits the same gross physical prop- erties as those previously reported in the literature. However, the distribution of half-cystine residues between the free sulf- hydryl and disulfide forms appears to be markedly different from that reported by Wallenfels et al. (25) for the enzyme isolated from the ML30 strain of E. coli. These investigators first suggested the existence of disulfide bonds between subunits since dissociation into small units could be obtained only after oxidation of cysteine residues. They have recently extended their studies through the use of mercuribenzoate titration and have concluded that, in their preparations, approximately 46 of 84 to 88 half-cystine residues can be titrated with this reagent in 8 M urea. They also have reported that only 46 cysteine residues are suscept,ible to alkylation with iodoacetamide in 3 M guanidine. Their experiments do not allow differentiation between “buried” sulfhydryl groups and those present in disulfide linkage which might be reduced by prior reduction with mercaptoethanol.7

A fractionation procedure has been developed which permits the isolation of relatively large quantities of fi-galactosidase from cells of Escherichia coli in a high state of purity as judged by a number of physical and immunological procedures. The major innovations in the present procedure involve the use of gel filtration through Sephadex G-200 and final purification on columns of diethylaminoethyl-Sephadex. Qualitative esamina- tion of homogeneity by polyacrylamide gel electrophoresis has been of special value in monitoring the efficacy of individual steps during fractionation. The final product from cells of E. coli Kl2 has a molecular weight in the neighborhood of 540,000, and this value is not reduced at high dilutions. Amino acid analysis indicates the presence of approximately 1,170 amino acid residues per quarter molecule, and together with studies on the suscepti- bility of half-cystine residues to alkylation, suggests that all of the latter residues exist in the sulfhydryl form. Approximately half of the sulfhydryl groups are reactive to iodoacetic acid in the native molecule without loss of enzymic activity.

11 cknowledgments -We would like to acknowledge the excellent technical assistance of Mrs. Lila Corley. We would also like to thank Dr. Frank J. Reithel for a generous gift of purified p- galactosidase during the early period of this work.

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Another point of departure appears to be in the number of -SH groups that can be titrated without loss in enzyme activity. Wallenfels et al. (25) have reported that mercuribenzoate reacts with only 12.8 -SH groups before loss in activity occurs whereas the studies reported here lead to the conclusion that approxi- mately half of a total of 78 -SH groups may be reacted without inactivation.

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These differences may reflect dissimilarities in the states of the enzyme as prepared by the respective methods. Wallenfels et al. have isolated the protein in the absence of reducing agents, whereas the enzyme preparation reported here involved the use of mercaptoethanol as a protective agent throughout the proce- dure. Galactosidase assayed in the normal fashion without mercaptoethanol, yields consistently lower values than those obtained in the presence of this substance. Furthermore, galactosidase which has been isolated in the presence of mer- captoethanol is much more heat-stable in its presence than in its

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7 It is important to note, that Weber, Sund, and Wallenfels have recently fragmented galactosidase into its smallest subunits without prior reduction of disulfide bridges with a formic acid- acetic acid solvent system (26).

BALBINDER, E., AND PREER, J. R., JR., J. Gen. Microbial., 21, 156 (1959).

15. PREER, J. R., AND PREER, L. B., J. Protozoal., 6, 88 (1959). 16. PREER, J. R., JR., J. Immunol., 77, 52 (1956). 17. SCHACHMAN, H. K., in S. P. COLOWICK AND N. 0. KAPLAN

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Gary R. Craven, Edward Steers, Jr. and Christian B. Anfinsen K12Escherichia coli

-Galactosidase of βPurification, Composition, and Molecular Weight of the

1965, 240:2468-2477.J. Biol. Chem. 

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