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A Rapid PurificationProcedure for L-Asparaginase from VibrioSuccinogenesAbraham Abuchowski a b , David Kafkewitz a b &Frank F. Davis a ba Department of Biochemistry, RutgersUniversity, New Brunswick, N. J., 08903b Department of Zoology and Physiology, RutgersUniversity, Newark, N. J., 07102Version of record first published: 05 Dec 2006.

To cite this article: Abraham Abuchowski , David Kafkewitz & Frank F.Davis (1979): A Rapid Purification Procedure for L-Asparaginase from VibrioSuccinogenes, Preparative Biochemistry, 9:3, 205-211

To link to this article: http://dx.doi.org/10.1080/00327487908061685

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PREPARATIVE BIOCHEMISTRY, 9(3), 205-211 (1979)

A RAPID PURIFICATION PROCEDURE FOR

L-ASPARAGINASE FROM VIBRIO SUCCINOGENES

Abraham Abuchowski, David Kafkewitz, and Frank F. Davis

Department of Biochemistry, Rutgers University New Brunswick, N. J. 08903, and

Department of Zoology and Physiology, Rutgers University Newark, N. J. 07102

ABSTRACT

A simple procedure has been developed for the purification

of L-asparaginase from Vibrio succinogenes. Only two steps of

ion-exchange chromatography are required. A higher yield and

higher specific activity are obtained than previously reported.

INTRODUCTION

Asparaginase (EC 3.5.1.1) preparations from several

~ourcesl-~ have been tested for antitumor activity since 1961,

when Broome first proposed that this enzyme was responsible for

the antilymphoma activity of guinea pig serum . Recently, an

asparaginase from Vibrio succinogenes6 has been isolated and

characterized. This organism produces large quantitites of an

asparaginase which is specific for L-asparagine and also appears

to possess antitumor These properties suggest

potential use for this enzyme in cancer therapy.

5

205

Copkright 0 1979 hy MdrLel Dekker. I n L Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system. w-fhout permission in writing from the publisher

All Rights Reserved

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206 ABUCHOWSKI, KAFKFMITZ, ANTI DAVIS

The published purification procedure6 involves a number of

precipitations followed by three steps of column chromatography.

A simple, rapid purification procedure would have obvious value

in the large scale production of this enzyme for therapeutic

evaluation. We report here a new purification procedure in-

volving only two steps of ion-exchange chromatography.

MATERIALS AND METHODS

CM-BioGel A and DEAE-BioGel A are products of BIO-RAD Lab-

oratories. Deoxyribonuclease was purchased from Calbiochem.

L-asparagine monohydrate was supplied by Aldrich Chemical Co.

L-aspartyl-B-hydroxamic acid and bovine serum albumin were ob-

tained from Nutritional Biochemicals Co. All other reagents

were from Fisher Scientific Co.

Vibrio succinogenes was grown in VSF medium' modified by

the addition, before autoclaving of cysteine-HC1 to a concen-

tration of 0.1%, and after autoclaving by the addition of

MgC12'6H 0 to a concentration of 0.01%. This 1. succinogenes

has been deposited in the culture collections of the Virginia

Polytechnic Institute Anaerobe Laboratory (V.P.I. 10659), and

the American Type Culture Collection (ATCC 29543).

2

Assay Methods. The L-aspartyl-8-hydroxamic acid assay of

Frohwein et a1.' was employed as a qualitative method to

quickly locate column fractions containing enzymic activity.

For specific activities, a modification of the procedure of

Meister et a1.l' was employed.

- M Tris-HC1, pH 8.0, was warmed to 37 C and the reaction was

Enzyme, 20 pg in 0.5 ml of b.05

0

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L-ASPARAGINASE FROM VIBRIO SUCCINOGENES 207

started by the addition of 0.5 ml 0.08 M L-asparagine. At 30

sec intervals, 0.1 ml aliquots were removed and pipetted into

tubes containing 0.1 ml 10% trichloroacetic acid. All tubes,

including a blank, were brought to 3.5 ml with water, followed

by the addition of 0.5 ml Nessler's reagent. One unit of as-

paraginase activity is defined as the liberation of 1 pmole NH3

per minute at 37O~.

Protein was determined by the biuret method using bovine

serum albumin as the standard. Sodium dodecyl sulfate diyc gel

electrophoresis was carried out by the procedure of Laerrzdi and

Favre . 11

EXPERIMENTAL PROCEDURE AND RESULTS

The purification was carried out at 4OC. All buffers were

prepared at room temperature. Packed, frozen cells (22 9) were

thawed in an equal volume of 0.01 ! Tris-HC1, pH 6.8, containing

1 mg deoxyribonuclease. The cell suspension was sonicated four

times for 30 sec each time at 90 watts with a Branson Sonifier.

The suspension was cooled for 30 sec in an ice bath between

sonications. Cell debris was removed by centrifugation at

40,000 x g for 30 min at 4OC.

buffer and the sonication and centrifugation procedures repeated.

The resulting pellet was washed with buffer. The two super-

natant liquids and wash were combined and centrifuged at 150,000

x g for 90 min at 4OC.

contained a total of 19,200 units of activity and 1940 mg pro-

tein, was dialyzed against two changes of 4 liters of 0.01 !

Tris-HC1, pH 6.8. The protein solution was applied to a

The pellet was resuspended in

The supernatant fluid (190 ml), which

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208 ABUCHOWSKI, KAFKEWITZ, AND DAVIS

CM-BioGel A column (2 .6 x 30 cm) previously equilibrated with

0.01 fl Tris-HC1, pH 6.8, at 33 ml/hr, followed immediately by a

200 ml buffer wash. A 1000 ml, 0-1.0 fl NaCl linear gradient in 0.01 fl Tris-HC1, pH 6.8, was applied to the column at the same

flow rate. Each fraction ( 5 ml) was tested for activity using

L-aspartyl-8-hydroxamic acid as the substrate. The enzyme was

eluted from the column, together with other proteins, at a salt

concentration of approximately 0.25 fl (Fig. 1, left). Fractions

containing enzymatic activity were pooled and assayed.

proximate 11-fold purification resulted, with a 73% yield.

An ap-

The enzyme solution was dialyzed against 5 volumes of 0.01

- M Tris-HC1, pH 9.0, using an Amicon Thin-Channel Ultrafilter

equipped with a PM-30 membrane. Dialysis removes some

minor contaminants and produces approximately a 10% increase in

specific activity. Fifty-five ml of enzyme solution containing

14,000 units of activity were applied to a 2.6 x 30 cm DEAE-

BioGel A column equilibrated with 0.01 E Tsis-HC1, pH 9.0, at

the rate of 33 ml/hr.

1000 ml, 0-0.3 E NaCl linear gradient in 0.01 E Tris-HC1, pH

9.0 (Fig. 1, right). Fractions containing activity were pooled,

dialyzed and concentrated in 0.05 E sodium phosphate at pH 7.3.

A yield of 69% was obtained with 23.5-fold purification over

the initial extract.

of 233 units/mg protein, somewhat higher than the specific

A 200 ml buffer wash was followed by a

The final product had a specific activity

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L-ASPARAGINASE FROM VIBRIO SUCCINOGENES

m - 6 2

- 4 5

- 2 $

\ v) c

w v)

U

a U

v) a

- 0

209

30

40

5 0

50

w " z 4

; 6 0

I YI * 4

a 70

."

80

90

100

F R A C T I O N NUMBER

.

FRACTION NUMBER

FIGURE 1

Chromatographic purification of asparaginase on CM-BioGel A (left) and DEAE-BioGel A (right). Asparaginase activity coincided with the first peak of the CM-BioGel A column. Fractions 69-74 were saved. Asparaginase activity coincided with the peak of the CM-BioGel A column. Fractions 26-31 were pooled. protein/ml.

An A280 reading of 1.0 corresponds to 4.0 mg Shaded regions indicate asparaginase activity.

6 activity of 200 units/mg protein reported by Distasio et al.

for their preparation.

acrylamide gel (Fig. 2,D).

It appeared as a single band on SDS

This procedure substantially decreases the time required

for purification of the enzyme, which is undergoing evaluation

for antitumor activity.

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210 ABUCHOWSKI, KAFKEWITZ, AND DAVIS

FIGURE 2

SDS disc gel electrophoresis of samples from various steps in the purification of v. succinogenes asparaginase. A, cell extract; B, pooled fractions from the CM-BioGel A column; C, the same pooled fractions following ultrafiltration; D, pooled fractions from the DEAE-BioGel A column.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Charles and

Johanna Busch Memorial Fund and by Research Grant GM-20946

from the National Institutes of Health.

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L-ASPARAGINASE FROM VIBRIO SUCC~NOGENES 211

1.

2

3.

4.

5.

6.

7.

8.

9.

10.

11.

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