Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BLUE SEPHAROSE*:
A REUSABLE AFFINITY
MEDIUM FOR PURIFICATION
CHROMATOGRAPHY
OF ALCOHOL DEHYDROGENASE
Geraldine E. Lamkin and E. E. King Cotton Physiology Laboratory Agricultural Research Service U.S. Department of Agriculture Stoneville, Mississippi 38776
Received July 14,1976 SUMMARY: The separation of alcohol dehydrogenase from crude cottonseed extracts is accomplished in a single step by affinity chromatography. Blue Sepharose 6B effectively binds the enzyme, and elution is effected with NAD. The enzyme thus purified has a specific activity forty times higher than the original extract, and yields two closely-spaced bands with gel electrophoresis. The column is reusable for many separations without loss of efficiency. Blue Sepharose
6B, a dye-coupled
cross-linked
agarose,
has been
shown to be an effective binding matrix for kinases and dehydrogenases. Selective
elution may be accomplished
concentrations alcohol
and pH.
dehydrogenase
Elution buffer recovering
Blue Sepharose (E.C.I.I.I.I)
by careful
6B has been used to separate
and other enzymes
for alcohol dehydrogenase
55% of the activity.
selection of cofactor
contained
The specific
enzyme was 31X that of the crude extract
application
extracts.
5 to i0 mM NAD,
activity of the purified
(i).
We have applied the technique of purification crude cottonseed
from yeast.
This paper reports
by Blue Sepharose
to
the details of our
and the results obtained with its use on the alcohol
dehydrogenase
of cotton
seeds.
METHODS Cotton seeds (Gossypium hirsutum L.) were dehulled in a disc mill. Hulls and fines were removed by air classification, and the remaining
* Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
560
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
kernels w e r e stored d e s i c c a t e d at -50 ° . The kernels w e r e g r o u n d in a knife m i l l for 15-30 seconds and b r u s h e d through a 60-mesh Screen. This fine m e a l was h o m o g e n i z e d and w a s h e d e x h a u s t i v e l y with -20 ° acetone to remove lipid material. F o l l o w i n g a final w a s h w i t h -20 ° ether, the meal was dried at r o o m temperature. Soluble proteins were e x t r a c t e d f r o m 0.5 g meal with 7.5 ml cold water and c e n t r i f u g e d at 7700 x g for 20 min. at 5 ° . A p p r o x i m a t e l y 0.5 g dry Blue Sepharose, h y d r a t e d o v e r n i g h t in d i s t i l l e d water, y i e l d e d a p a c k e d column 0.8 x 12 cm. The column was e q u i l i b r a t e d with starting buffer c o n s i s t i n g of 20 m M Tris HCI, pH 6.4, 5 m M MgCI2, 0.4 m M EDTA, and 1 m M 2-mercaptoethanol. The column, all solutions, and fractions were m a i n t a i n e d at 5 °. The water extract, c o n t a i n i n g units of alcohol d e h y d r o g e n a s e per buffer. After 4 ml of the diluted the starting b u f f e r was allowed to remove u n b o u n d sample material.
3.2 mg p r o t e i n and 0.8 international ml, was d i l u t e d i:i w i t h starting extract was loaded onto the column, flow o v e r n i g h t at 0.12 m l / m i n to
E l u t i o n of alcohol d e h y d r o g e n a s e was a t t e m p t e d with various c o n c e n t r a t i o n s of N A D in starting buffer. Flow rate was 0.12 ml/min; successive 0.6 ml fractions were c o l l e c t e d until alcohol d e h y d r o g e n a s e eluti0n was complete. F o l l o w i n g elution, the column was w a s h e d with 5 ml 2 M NaCl in starting buffer, and e q u i l i b r a t e d w i t h 25 ml starting b u f f e r for reuse. Alcohol d e h y d r o g e n a s e a c t i v i t y was d e t e r m i n e d s p e c t r o p h o t o m e t r i cally by m e a s u r i n g the increase in a b s o r b a n c e at 340 nm due to r e d u c t i o n of NAD with ethanol substrate (2). Enzyme activities are e x p r e s s e d in international units. P r o t e i n content was estimated by a Lowry procedure, m o d i f i e d for use in the p r e s e n c e of interfering m a t e r i a l s such as Tris (3). E l e c t r o p h o r e s i s of crude extracts and alcohol d e h y d r o g e n a s e - a c t i v e fractions was a c c o m p l i s h e d using 2.5-27% linear gradient p o l y a c r y l a m i d e gel slabs in 0.065 M T r i s - b o r a t e buffer, pH 9.0. P r o t e i n bands in the gels were d e t e c t e d with Coomassie b r i l l i a n t blue R-250 (4); alcohol d e h y d r o g e n a s e a c t i v i t y in the gels was indicated by n i t r o - b l u e tetraz o l i u m (5).
RESULTS
AND DISCUSSION
The Blue S e p h a r o s e from crude
unbuffered
essentially
complete,
starting
buffer.
precisely
column
water
determined,
dehydrogenase,
Initial
attempts
when
(pH 6.5).
appears
dehydrogenase
Binding
was
the extract was d i l u t e d
the point of column
the m a x i m u m
0.5 g dry Blue Sepharose alcohol
extracts
however,
Although
failed to b i n d alcohol
capacity
overload
at elution were
561
was not
of a column p r e p a r e d
to be a p p r o x i m a t e l y
or twice the amount
i:i with
3 units
used in these
successful
using
of
studies.
5 mM NAD
from
Vol. 72, No. 2, 1976
Table i.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
R e c o v e r y and P u r i f i c a t i o n Blue S e p h a r o s e 6B
Elution S t a r t i n g Buffer p l u s
of Alcohol
Dehydrogenase
with
Recovery enzyme units
mg protein
specific activity
per cent recovered
purification
1 mM N A D
0.75
0.075
9.99
51%
43X
2.5 m M NAD
0.61
0.064
9.52
48%
40X
5 m M NAD
0.74
0.070
10.68
50%
45X
0-i m M NAD g r a d i e n t
0.51
0.050
10.14
30%
42X
0-5 m M NAD g r a d i e n t
0.75
0.073
10.25
46%
43X
Sample loaded onto c o l u m n c o n t a i n e d 1.5 + 0.2 units of enzyme a c t i v i t y and 6.4 + 0.7 mg p r o t e i n in 4.0 ml, specific a c t i v i t y 0.239 + 0.003.
in s t a r t i n g buffer.
Purification
the enzyme was eluted w i t h gradient
of 0-5 m M NAD
and r e c o v e r y were c o m p a r a b l e w h e n
i, 2.5,
(Table i).
and 5.0 m M NAD, However,
r e c o v e r y was
the enzyme was eluted w i t h a linear gradient Elution patterns in F i g u r e
i.
Monitoring
NAD concentration,
dehydrogenase
the a b s o r b a n c e
in fact,
and p r o t e i n are shown
at 280 nm i n d i c a t e d relative
large enough to detect.
T e s t i n g the fractions
a c t i v i t y and for p r o t e i n
content r e v e a l e d
that the enzyme f o l l o w e d the leading edge of the NAD. b o t h enzyme activity
p e a k e d near fraction
7.
and p r o t e i n
the 0-i m M NAD g r a d i e n t peaks
However,
near
difference
c o n t e n t are sharply
the elution p a t t e r n by
fraction 13, and is not as sharply
as the e l u t i o n p a t t e r n by 1 m M NAD.
corresponding
For the 1 m M
The 0 to 5 m M N A D elutions all gave p a t t e r n s
s i m i l a r to the 1 m M NAD elution.
defined
at this wavelength.
the p e a k w h i c h the p r o t e i n w o u l d have given
for a l c o h o l d e h y d r o g e n a s e
NAD elution,
lowered w h e n
of 0-i mM.
since the NAD has some a b s o r b a n c e
The NAD p e a k obscures if it were,
for alcohol
or w i t h a linear
Table I shows a
in r e c o v e r y w i t h the 0 to 1 m M NAD
gradient.
562
VoI. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
'~'! 4
alcohol dehydrogenase
0.20 A
2.0
1 mM NAD E LUTION
"~/
E
>- 0.15
/ ~
"< A280 nm 1.0
~ 0.10
0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BLUE SEPHAROSE*:
A REUSABLE AFFINITY
MEDIUM FOR PURIFICATION
CHROMATOGRAPHY
OF ALCOHOL DEHYDROGENASE
Geraldine E. Lamkin and E. E. King Cotton Physiology Laboratory Agricultural Research Service U.S. Department of Agriculture Stoneville, Mississippi 38776
Received July 14,1976 SUMMARY: The separation of alcohol dehydrogenase from crude cottonseed extracts is accomplished in a single step by affinity chromatography. Blue Sepharose 6B effectively binds the enzyme, and elution is effected with NAD. The enzyme thus purified has a specific activity forty times higher than the original extract, and yields two closely-spaced bands with gel electrophoresis. The column is reusable for many separations without loss of efficiency. Blue Sepharose
6B, a dye-coupled
cross-linked
agarose,
has been
shown to be an effective binding matrix for kinases and dehydrogenases. Selective
elution may be accomplished
concentrations alcohol
and pH.
dehydrogenase
Elution buffer recovering
Blue Sepharose (E.C.I.I.I.I)
by careful
6B has been used to separate
and other enzymes
for alcohol dehydrogenase
55% of the activity.
selection of cofactor
contained
The specific
enzyme was 31X that of the crude extract
application
extracts.
5 to i0 mM NAD,
activity of the purified
(i).
We have applied the technique of purification crude cottonseed
from yeast.
This paper reports
by Blue Sepharose
to
the details of our
and the results obtained with its use on the alcohol
dehydrogenase
of cotton
seeds.
METHODS Cotton seeds (Gossypium hirsutum L.) were dehulled in a disc mill. Hulls and fines were removed by air classification, and the remaining
* Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
560
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
kernels w e r e stored d e s i c c a t e d at -50 ° . The kernels w e r e g r o u n d in a knife m i l l for 15-30 seconds and b r u s h e d through a 60-mesh Screen. This fine m e a l was h o m o g e n i z e d and w a s h e d e x h a u s t i v e l y with -20 ° acetone to remove lipid material. F o l l o w i n g a final w a s h w i t h -20 ° ether, the meal was dried at r o o m temperature. Soluble proteins were e x t r a c t e d f r o m 0.5 g meal with 7.5 ml cold water and c e n t r i f u g e d at 7700 x g for 20 min. at 5 ° . A p p r o x i m a t e l y 0.5 g dry Blue Sepharose, h y d r a t e d o v e r n i g h t in d i s t i l l e d water, y i e l d e d a p a c k e d column 0.8 x 12 cm. The column was e q u i l i b r a t e d with starting buffer c o n s i s t i n g of 20 m M Tris HCI, pH 6.4, 5 m M MgCI2, 0.4 m M EDTA, and 1 m M 2-mercaptoethanol. The column, all solutions, and fractions were m a i n t a i n e d at 5 °. The water extract, c o n t a i n i n g units of alcohol d e h y d r o g e n a s e per buffer. After 4 ml of the diluted the starting b u f f e r was allowed to remove u n b o u n d sample material.
3.2 mg p r o t e i n and 0.8 international ml, was d i l u t e d i:i w i t h starting extract was loaded onto the column, flow o v e r n i g h t at 0.12 m l / m i n to
E l u t i o n of alcohol d e h y d r o g e n a s e was a t t e m p t e d with various c o n c e n t r a t i o n s of N A D in starting buffer. Flow rate was 0.12 ml/min; successive 0.6 ml fractions were c o l l e c t e d until alcohol d e h y d r o g e n a s e eluti0n was complete. F o l l o w i n g elution, the column was w a s h e d with 5 ml 2 M NaCl in starting buffer, and e q u i l i b r a t e d w i t h 25 ml starting b u f f e r for reuse. Alcohol d e h y d r o g e n a s e a c t i v i t y was d e t e r m i n e d s p e c t r o p h o t o m e t r i cally by m e a s u r i n g the increase in a b s o r b a n c e at 340 nm due to r e d u c t i o n of NAD with ethanol substrate (2). Enzyme activities are e x p r e s s e d in international units. P r o t e i n content was estimated by a Lowry procedure, m o d i f i e d for use in the p r e s e n c e of interfering m a t e r i a l s such as Tris (3). E l e c t r o p h o r e s i s of crude extracts and alcohol d e h y d r o g e n a s e - a c t i v e fractions was a c c o m p l i s h e d using 2.5-27% linear gradient p o l y a c r y l a m i d e gel slabs in 0.065 M T r i s - b o r a t e buffer, pH 9.0. P r o t e i n bands in the gels were d e t e c t e d with Coomassie b r i l l i a n t blue R-250 (4); alcohol d e h y d r o g e n a s e a c t i v i t y in the gels was indicated by n i t r o - b l u e tetraz o l i u m (5).
RESULTS
AND DISCUSSION
The Blue S e p h a r o s e from crude
unbuffered
essentially
complete,
starting
buffer.
precisely
column
water
determined,
dehydrogenase,
Initial
attempts
when
(pH 6.5).
appears
dehydrogenase
Binding
was
the extract was d i l u t e d
the point of column
the m a x i m u m
0.5 g dry Blue Sepharose alcohol
extracts
however,
Although
failed to b i n d alcohol
capacity
overload
at elution were
561
was not
of a column p r e p a r e d
to be a p p r o x i m a t e l y
or twice the amount
i:i with
3 units
used in these
successful
using
of
studies.
5 mM NAD
from
Vol. 72, No. 2, 1976
Table i.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
R e c o v e r y and P u r i f i c a t i o n Blue S e p h a r o s e 6B
Elution S t a r t i n g Buffer p l u s
of Alcohol
Dehydrogenase
with
Recovery enzyme units
mg protein
specific activity
per cent recovered
purification
1 mM N A D
0.75
0.075
9.99
51%
43X
2.5 m M NAD
0.61
0.064
9.52
48%
40X
5 m M NAD
0.74
0.070
10.68
50%
45X
0-i m M NAD g r a d i e n t
0.51
0.050
10.14
30%
42X
0-5 m M NAD g r a d i e n t
0.75
0.073
10.25
46%
43X
Sample loaded onto c o l u m n c o n t a i n e d 1.5 + 0.2 units of enzyme a c t i v i t y and 6.4 + 0.7 mg p r o t e i n in 4.0 ml, specific a c t i v i t y 0.239 + 0.003.
in s t a r t i n g buffer.
Purification
the enzyme was eluted w i t h gradient
of 0-5 m M NAD
and r e c o v e r y were c o m p a r a b l e w h e n
i, 2.5,
(Table i).
and 5.0 m M NAD, However,
r e c o v e r y was
the enzyme was eluted w i t h a linear gradient Elution patterns in F i g u r e
i.
Monitoring
NAD concentration,
dehydrogenase
the a b s o r b a n c e
in fact,
and p r o t e i n are shown
at 280 nm i n d i c a t e d relative
large enough to detect.
T e s t i n g the fractions
a c t i v i t y and for p r o t e i n
content r e v e a l e d
that the enzyme f o l l o w e d the leading edge of the NAD. b o t h enzyme activity
p e a k e d near fraction
7.
and p r o t e i n
the 0-i m M NAD g r a d i e n t peaks
However,
near
difference
c o n t e n t are sharply
the elution p a t t e r n by
fraction 13, and is not as sharply
as the e l u t i o n p a t t e r n by 1 m M NAD.
corresponding
For the 1 m M
The 0 to 5 m M N A D elutions all gave p a t t e r n s
s i m i l a r to the 1 m M NAD elution.
defined
at this wavelength.
the p e a k w h i c h the p r o t e i n w o u l d have given
for a l c o h o l d e h y d r o g e n a s e
NAD elution,
lowered w h e n
of 0-i mM.
since the NAD has some a b s o r b a n c e
The NAD p e a k obscures if it were,
for alcohol
or w i t h a linear
Table I shows a
in r e c o v e r y w i t h the 0 to 1 m M NAD
gradient.
562
VoI. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
'~'! 4
alcohol dehydrogenase
0.20 A
2.0
1 mM NAD E LUTION
"~/
E
>- 0.15
/ ~
"< A280 nm 1.0
~ 0.10
0
