This article was downloaded by: [University of Illinois Chicago] On: 17 April 2013, At: 05:42 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
A Rapid Purification Procedure for LAsparaginase from Vibrio Succinogenes Abraham Abuchowski Frank F. Davis
a b
, David Kafkewitz
a b
&
a b
a
Department of Biochemistry, Rutgers University, New Brunswick, N. J., 08903 b
Department of Zoology and Physiology, Rutgers University, Newark, N. J., 07102 Version 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 Vibrio Succinogenes, Preparative Biochemistry, 9:3, 205-211 To link to this article: http://dx.doi.org/10.1080/00327487908061685
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/ terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
PREPARATIVE BIOCHEMISTRY, 9(3), 205-211 (1979)
A
RAPID PURIFICATION PROCEDURE FOR
L-ASPARAGINASE FROM VIBRIO SUCCINOGENES
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
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 5 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.
205 Copkright 0 1979 hy MdrLel Dekker. I n L All Rights Reserved 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
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
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
evaluation. We report here a new purification procedure involving only two steps of ion-exchange chromatography. MATERIALS AND METHODS CM-BioGel A and DEAE-BioGel A are products of BIO-RAD Laboratories.
Deoxyribonuclease was purchased from Calbiochem.
L-asparagine monohydrate was supplied by Aldrich Chemical Co. L-aspartyl-B-hydroxamic acid and bovine serum albumin were obtained 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-HC1to a concentration of 0.1%, and after autoclaving by the addition of MgC12'6H 0 to a concentration of 0.01%. 2
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). Assay Methods. Frohwein et a1.'
The L-aspartyl-8-hydroxamic acid assay of
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'
M Tris-HC1, pH
was employed.
Enzyme, 20 pg in 0.5 ml of b.05 0
8.0, was warmed to 37 C and the reaction was
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,
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
by the addition of 0.5 ml Nessler's reagent.
followed
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 11 Favre
. 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.
The pellet was resuspended in
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.
The supernatant fluid (190 ml), which
contained a total of 19,200 units of activity and 1940 mg protein, was dialyzed against two changes of 4 liters of 0.01 Tris-HC1, pH 6.8.
The protein solution was applied to a
!
ABUCHOWSKI, KAFKEWITZ, AND DAVIS
208 CM-BioGel 0.01
A
column ( 2 . 6 x 30 cm) previously equilibrated with
fl Tris-HC1, pH
6.8, at 33 ml/hr, followed immediately by a
200 ml buffer wash.
0.01
fl Tris-HC1, pH
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
flow rate.
A
1000 ml, 0-1.0 fl NaCl linear gradient in
6.8,
was applied to the column at the same
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.
An
ap-
proximate 11-fold purification resulted, with a 73% yield. 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 the rate of 3 3 ml/hr. 1000 ml, 0-0.3
A
9.0, at
200 ml buffer wash was followed by a
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 A
E Tsis-HC1, pH
E sodium phosphate at pH 7.3.
yield of 69% was obtained with 23.5-fold purification over
the initial extract.
The final product had a specific activity
of 233 units/mg protein, somewhat higher than the specific
.
L-ASPARAGINASE FROM VIBRIO SUCCINOGENES
30
40
5
209
0
50
"z4 w
;
60
m
I
*4
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
YI
a
-6
2
\
70
v)
."
- 4 80
c 5
v) w
U
-2
90
$ a U a v)
-0
100
FRACTION NUMBER
F R A C T I O N 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. An A280 reading of 1.0 corresponds to 4.0 mg protein/ml. Shaded regions indicate asparaginase activity.
activity of 200 units/mg protein reported by Distasio et al. for their preparation.
6
It appeared as a single band on SDS
acrylamide gel (Fig. 2,D). This procedure substantially decreases the time required for purification of the enzyme, which is undergoing evaluation for antitumor activity.
ABUCHOWSKI, KAFKEWITZ, AND DAVIS
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
210
FIGURE 2 SDS disc gel electrophoresis of samples from various succinogenes asparaginase. A, steps in the purification of 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.
v.
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.
L-ASPARAGINASE FROM VIBRIO SUCC~NOGENES
211
REFERENCES
1. J. Roberts, G. Burson and J. M. Hill, J. Bacteriol., 2117-2123 2
(1968).
P. E. Joner, T. Kristiansen and M. Einarsson, Biochim.
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
Biophys. Acta, 3.
E,
327, 146-156
(1973).
H. A. Whelan and J. C. Wriston, Jr., Biochim. Biophys. Acta, 365, -
212-222
(1974)
. 2, 2119-2123
4.
J. Roberts, J. Biol. Chem.,
5.
J. D. Broome, Yature,
6.
J. A. Distasio, R. A. Niederman, D. Kafkewitz and G. Goodman, J. Biol. Chem.,
7.
191,1114-1115
(1976).
(1961).
=,
6929-6933.
D. Kafkewitz and D. Goodman, Appl. Microbiol., 27,206-209 (1974).
8.
D. Kafkewitz, Appl. Microbiol.,
9.
Y.
Z.
2,121-122
(1975).
Frohwein, M. Friedman, J. Riezer and N. Grossowicz,
Nature,
9, 158-159
(1971).
,
10. A. Meister, L. Levintow, R. E. Greenfield and P. A. AbendSchein, J. Biol. Chem. , 215,441-460 11.
(1955).
U . K. Laemmli and M. Favre, J. Mol. Biol., (1973).
80, 575-599
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
A Rapid Purification Procedure for LAsparaginase from Vibrio Succinogenes Abraham Abuchowski Frank F. Davis
a b
, David Kafkewitz
a b
&
a b
a
Department of Biochemistry, Rutgers University, New Brunswick, N. J., 08903 b
Department of Zoology and Physiology, Rutgers University, Newark, N. J., 07102 Version 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 Vibrio Succinogenes, Preparative Biochemistry, 9:3, 205-211 To link to this article: http://dx.doi.org/10.1080/00327487908061685
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/ terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
PREPARATIVE BIOCHEMISTRY, 9(3), 205-211 (1979)
A
RAPID PURIFICATION PROCEDURE FOR
L-ASPARAGINASE FROM VIBRIO SUCCINOGENES
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
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 5 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.
205 Copkright 0 1979 hy MdrLel Dekker. I n L All Rights Reserved 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
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
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
evaluation. We report here a new purification procedure involving only two steps of ion-exchange chromatography. MATERIALS AND METHODS CM-BioGel A and DEAE-BioGel A are products of BIO-RAD Laboratories.
Deoxyribonuclease was purchased from Calbiochem.
L-asparagine monohydrate was supplied by Aldrich Chemical Co. L-aspartyl-B-hydroxamic acid and bovine serum albumin were obtained 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-HC1to a concentration of 0.1%, and after autoclaving by the addition of MgC12'6H 0 to a concentration of 0.01%. 2
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). Assay Methods. Frohwein et a1.'
The L-aspartyl-8-hydroxamic acid assay of
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'
M Tris-HC1, pH
was employed.
Enzyme, 20 pg in 0.5 ml of b.05 0
8.0, was warmed to 37 C and the reaction was
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,
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
by the addition of 0.5 ml Nessler's reagent.
followed
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 11 Favre
. 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.
The pellet was resuspended in
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.
The supernatant fluid (190 ml), which
contained a total of 19,200 units of activity and 1940 mg protein, was dialyzed against two changes of 4 liters of 0.01 Tris-HC1, pH 6.8.
The protein solution was applied to a
!
ABUCHOWSKI, KAFKEWITZ, AND DAVIS
208 CM-BioGel 0.01
A
column ( 2 . 6 x 30 cm) previously equilibrated with
fl Tris-HC1, pH
6.8, at 33 ml/hr, followed immediately by a
200 ml buffer wash.
0.01
fl Tris-HC1, pH
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
flow rate.
A
1000 ml, 0-1.0 fl NaCl linear gradient in
6.8,
was applied to the column at the same
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.
An
ap-
proximate 11-fold purification resulted, with a 73% yield. 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 the rate of 3 3 ml/hr. 1000 ml, 0-0.3
A
9.0, at
200 ml buffer wash was followed by a
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 A
E Tsis-HC1, pH
E sodium phosphate at pH 7.3.
yield of 69% was obtained with 23.5-fold purification over
the initial extract.
The final product had a specific activity
of 233 units/mg protein, somewhat higher than the specific
.
L-ASPARAGINASE FROM VIBRIO SUCCINOGENES
30
40
5
209
0
50
"z4 w
;
60
m
I
*4
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
YI
a
-6
2
\
70
v)
."
- 4 80
c 5
v) w
U
-2
90
$ a U a v)
-0
100
FRACTION NUMBER
F R A C T I O N 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. An A280 reading of 1.0 corresponds to 4.0 mg protein/ml. Shaded regions indicate asparaginase activity.
activity of 200 units/mg protein reported by Distasio et al. for their preparation.
6
It appeared as a single band on SDS
acrylamide gel (Fig. 2,D). This procedure substantially decreases the time required for purification of the enzyme, which is undergoing evaluation for antitumor activity.
ABUCHOWSKI, KAFKEWITZ, AND DAVIS
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
210
FIGURE 2 SDS disc gel electrophoresis of samples from various succinogenes asparaginase. A, steps in the purification of 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.
v.
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.
L-ASPARAGINASE FROM VIBRIO SUCC~NOGENES
211
REFERENCES
1. J. Roberts, G. Burson and J. M. Hill, J. Bacteriol., 2117-2123 2
(1968).
P. E. Joner, T. Kristiansen and M. Einarsson, Biochim.
Downloaded by [University of Illinois Chicago] at 05:42 17 April 2013
Biophys. Acta, 3.
E,
327, 146-156
(1973).
H. A. Whelan and J. C. Wriston, Jr., Biochim. Biophys. Acta, 365, -
212-222
(1974)
. 2, 2119-2123
4.
J. Roberts, J. Biol. Chem.,
5.
J. D. Broome, Yature,
6.
J. A. Distasio, R. A. Niederman, D. Kafkewitz and G. Goodman, J. Biol. Chem.,
7.
191,1114-1115
(1976).
(1961).
=,
6929-6933.
D. Kafkewitz and D. Goodman, Appl. Microbiol., 27,206-209 (1974).
8.
D. Kafkewitz, Appl. Microbiol.,
9.
Y.
Z.
2,121-122
(1975).
Frohwein, M. Friedman, J. Riezer and N. Grossowicz,
Nature,
9, 158-159
(1971).
,
10. A. Meister, L. Levintow, R. E. Greenfield and P. A. AbendSchein, J. Biol. Chem. , 215,441-460 11.
(1955).
U . K. Laemmli and M. Favre, J. Mol. Biol., (1973).
80, 575-599
