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Improved Isolation Procedures for the Purple Membrane of Halobacterium Halobium a

Brian M. Becher & Seph Y. Cassim

a

a

Department of Biophysics, The Ohio State University Columbus, Ohio, 43210 Published online: 06 Dec 2006.

To cite this article: Brian M. Becher & Seph Y. Cassim (1975): Improved Isolation Procedures for the Purple Membrane of Halobacterium Halobium, Preparative Biochemistry, 5:2, 161-178 To link to this article: http://dx.doi.org/10.1080/00327487508061568

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PREPARATIVE BIOCHEMISTRY, 5(2),

161-178 (1975)

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IMPROVED ISOLATION PROCEDURES FOR THE PURPLE MEMBRANE OF HALOBACTERIUM HALOBIUM Brian M . Becher and Joseph Y . Cassim Department of Biophysics The Ohio S t a t e U n i v e r s i t y Columbus, Ohio 43210 ABSTRACT Techniques f o r p u r i f y i n g t h e purple membrane of Halobacterium halobium a r e given.

This purple membrane contains a chromoprotein with

a r e t i n a l p r o s t h e t i c g r o u p similar t o rhodopsin, t h e chromoprotein found i n t h e v i s u a l systems of higher i n v e r t e b r a t e s and v e r t e b r a t e s .

The

described purple membrane i s o l a t i o n procedures y i e l d a h i g h l y p u r i f i e d p r e p a r a t i o n as determined by t r a n s m i t t i n g e l e c t r o n microscopy and g e l electrophoresis.

C r i t i c a l a n a l y s i s of t h e absorption s p e c t r a of t h e

purple membrane was a l s o employed t o e s t a b l i s h c r i t e r i a of p u r i t y f o r t h e preparation.

The v i s i b l e absorption s p e c t r a of t h e p u r i f i e d p u r p l e mem-

brane p r e p a r a t i o n i n b u f f e r was found t o have a maximum a t 559 nm which shifted t o

567 nm on l i g h t exposure.

No i n d i c a t i o n of any s p e c t r a l

p e r t u r b a t i o n a r i s i n g from bacterioruberin-containing membrane, t h e major contaminant i n purple membrane p r e p a r a t i o n s , was found.

Furthermore,

t h e r a t i o of p r o t e i n aromatic amino a c i d absorbance a t 280 nm t o chromophore absorbance at

567 nm w a s found t o be 1 . 5 i n light-exposed prepara-

t i o n s compared t o t h e previously r e p o r t e d r a t i o of ~

0 The . decrease ~

i n t h e value of t h i s r a t i o i s a l s o i n d i c a t i v e of an i n c r e a s e i n t h e p u r i t y of t h e purple membrane p r e p a r a t i o n .

161 Copyright 0 I 9 7 5 hy Marcel Dekker. Inc. All Rlghts Reserved. Neither this work iior any part may he reproduced or transmitted in any form or by any means. electronic or niechanical, including photocopying, microfilming, and recording,or by any information storage and retrieval system. without permission in writing from the publisher.

BECHER AND CASSIM

162

INTRODUCTION

Halobacteria halobium i s an extremely h a l o p h i l i c b a c t e y i a which grows optimally i n sodium c h l o r i d e concentrations of 4 M o r more.

When

t h e s a l t concentration i s lowered t o 0.5 M t h e b a c t e r i a c e l l s r u p t u r e

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due t o osmotic shock.’

Continued lowering o f t h e s a l t concentration

causes t h e c e l l envelope t o f u r t h e r d i s a s s o c i a t e u n t i l only one t y p e of membrane from t h e b a c t e r i a i s r e a d i l y sedimented a t c e n t r i f u g a l f o r c e s of 50,OOOg.

Transmitting e l e c t r o n microscopy of t h i s

membrane r e v e a l s round-to-oval i n diameter and 50A t h i c k .

purple-colored

s h e e t s of membrane approximately 0 . 5

In vivo, t h i s membrane i s

currently

b e l i e v e d t o be l o c a t e d i n t h e c e l l membrane of t h e bacterium.

2

The name purple membrane has been a p p l i e d t o t h e s e membrane s h e e t s because of t h e i r d i s t i n c t i v e c o l o r . 3

The membrane d e r i v e s i t s

purple c o l o r from a chromoprotein containing a r e t i n a l p r o s t h e t i c group similar t o rhodopsin, t h e chromoprotein found i n t h e v i s u a l system of

higher i n v e r t e b r a t e s and v e r t e b r a t e s .

In view of t h i s s i m i l a r i t y , t h e

name “bacteriorhodopsin“ has been a p p l i e d t o t h e p u r p l e membrane chromoprotein.3 It h a s been r e p o r t e d t h a t l i g h t p e r t u r b a t i o n of t h e dark-adapted purple membrane i n b u f f e r r e s u l t s i n a r e d s h i f t and an increase i n i n t e n s i t y of t h e v i s i b l e absorption maximum.4

Furthermore,

recent evidence has l e d t o t h e s p e c u l a t i o n t h a t t h e purple membrane m a y d i r e c t l y convert l i g h t energy i n t o a proton g r a d i e n t a c r o s s t h e

b a c t e r i a l membrane i n vivo by c y c l i c light-induced conformation changes i n i t s chromoprotein.

This g r a d i e n t may be used i n t h e s y n t h e s i s of

.

ATP 5 The major contaminant i n t h e p u r p l e membrane p r e p a r a t i o n c o n s i s t s of f r a w e n t s of a red-colored membrane containing t h e c a r a t e n o i d bacterioruberin.6

I n vivo, both t h e purple and r e d membranes a r e found

i n t h e b a c t e r i a c e l l envelope.

Additional contamination of t h e p u r p l e

ISOLATING PURPLE MEMBRANE OF H. halobium

163

membrane p r e p a r a t i o n r e s u l t s from "intracytoplasmic membranes" which a r e believed t o be collapsed gas vacuoles.

The d i f f i c u l t problem of removing

t h e intracytoplasmic membranes, which have approximately t h e same d e n s i t y

as t h e purple membranes, i s avoided by t h e use of t h e mutant s t r a i n

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Halobacterium halobium R which does n o t produce gas vacuoles. .-. 1 S e v e r a l s t u d i e s have described t h e i s o l a t i o n of purple membrane d i f f e r from t h o s e from Halobacterium h a l ~ b i u m . ~ ' ~ ' ~ Our ' ~ ' procedures ~. previously reported i n two ways.

F i r s t , c e r t a i n common i s o l a t i o n pro-

cedures are replaced by s i m i l a r but d i s t i n c t methods which y i e l d a highly pure p r e p a r a t i o n .

Second, a c r i t i c a l a n a l y s i s of t h e v i s i b l e

absorption s p e c t r a of t h e bacteriorhodopsin chromophore, and t h e p r o t e i n to-chromophore r a t i o as determined by absorbance a t 280 nm and 567 nm,

was performed.

These c r i t i c a l s p e c t r a l methods, i n a d d i t i o n t o e l e c t r o n

microscopy and g e l e l e c t r o p h o r e s i s s t u d i e s were employed i n an e f f o r t t o e s t a b l i s h c r i t e r i a of p r e p a r a t i o n p u r i t y . MATERIALS AND INSTRUMENTATION The mutant Halobacterium halobium R1 employed i n t h i s study w a s a generous g i f t of Dr. Walter S p e r l i n g ( I n s t i t u t f i r Neurobiologie, J c l i c h Germany).

One l i t e r of growth medium contained 250g N a C 1 , 2g K CL , log

anhydrous MgS04, 3g trisodium c i t r a t e , and l o g Oxoid peptone (Code L37 Flow Lab I n c . , Rockville of reagent grade.

, Maryland), All

s a l t s used i n t h e medium were

Antifoam A Spray (now Corning, Midland, Michigan) w a s

a l s o used i n t h e growth medium.

DNAase 1 ( a c t i v i t y approximately 1400

Kunitz u n i t s p e r mg p r o t e i n , Sigma Chemical Company, S t . Louis, Missouri) was used i n e x t r a c t i o n procedures.

Standard marker p r o t e i n s employed i n

e l e c t r o p h o r e s i s were bovine serum albumin (Sigma),pepsinogen (Worthington Biochemical Corporation, Freehold, New J e r s e y ) , bovine pancreas t r y p s i n (Sigma), sperm whale w o g l o b i n (Mann Research L a b o r a t o r i e s , New York,

BECHER AND CASSIM

164

New York), and lysozyme (Worthington).

E l e c t r o p h o r e t i c grade sodium

dodecyl s u l f a t e (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) and Coomassie b r i l l i a n t blue s t a i n (Si@pla) were a l s o employed.

A l l other

chemicals used i n e l e c t r o p h o r e s i s were of reagent grade.

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The i l l u m i n a t i o n of t h e c u l t u r e medium was measured by a photometer (Model 200 M , Photovolt Corporation, New York, New York) modified by an opal and density 2 f i l t e r t o make t h e meter cosine c o r r e c t a b l e .

The

photometer was c a l i b r a t e d with a , t u n g s t e n filament s t a n d a r d lamp. Oxygen concentrations i n t h e growth medium were measured by a custom designed oxygen e l e c t r o d e .

Centrifuges employed included t h e S o r v a l l

Superspeed RC2-B c e n t r i f u g e with GSA and SS-34 r o t o r s (Dupont I n s t r u ment Product Division, Newtown, Connecticut) and t h e Beckman p r e p a r a t i v e u l t r a c e n t r i f u g e L2-65B with SW-41 T i r o t o r (Beckman Instrument Incorporated, Palo A l t o , C a l i f o r n i a ) .

A Radiometer Type pm26 (Radiometer,

Copenhagen, Denmark) was used t o measure pH.

Absorption of t h e

b a c t e r i a c u l t u r e s was measured throughout t h e i r growth periods by a Varian Techtron W - v i s i b l e spectrophotometer Model 635 (Varian Instrument D i v i s i o n , Palo Alto, C a l i f o r n i a ) .

Absorption s p e c t r a of

of membrane preparations was measured by a C a r y Model 118C double beam recording spectrophotometer with s c a t t e r e d transmission accessory (Varian).

A p a i r of o p t i c a l l y matched r e c t a n g u l a r absorption c e l l s with

fused s i l i c a windows ( F y r o c e l l Manufacturing Company, Westwood, New J e r s e y ) was employed in t h e spectrophotometric measurements.

A Zeiss

EM 9s e l e c t r o n microscope (Carl Zeiss Incorporated, New York, New York) w a s employed i n t h e e l e c t r o n microscopic s t u d i e s .

Disc g e l e l e c t r o -

phoresis was performed with a Bio-Rad e l e c t r o p h o r e s i s c e l l Model 300 (Bio-Rad Laboratories, Richmond, C a l i f o r n i a ) ,

Destaining w a s accomp-

l i s h e d with a d e s t a i n e r and d e s t a i n e r power supply ( I n s t r u m e n t a t i o n S p e c i a l i t y Company, Lincoln, Nebraska).

ISOLATING PURPLE MEMBRANE OF H. halobium

165

METHODS Growth of C e l l s .

Halobacteria halobium R

1

were Frown i n f r e s h c u l t u r e

media which was prepared by methods previously r e p ~ r t e d . ~ C e l l s were grown i n f o u r l i t e r q u a n t i t i e s which were innoculated with 300 m l of

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c u l t u r e a t t h e s t a t i o n a r y growth phase.

A t t h e s t a t i o n a r y growth

phase, using t h e c u l t u r e medium as a r e f e r e n c e , t h e b a c t e r i a c u l t u r e had an abso-bance of 2.2 at 560 nm.

The f o u r l i t e r c u l t u r e s were

grown i n 20 l i t e r g l a s s carboys supported i n a h o r i z o n t a l p o s i t i o n . Four h0 w a t t f l u o r e s c e n t l i g h t s (Sylvania L i f e l i n e F40CW) were placed d i r e c t l y above t h e carboys and f o u r d i r e c t l y below.

The illuminance

of t h e c u l t u r e s through t h e c l e a r g l a s s carboys (luminous t r a n s m i t t a n c e approximately 0 . 9 ) was 2,500 lumens p e r f o o t 2 (foot-candles) from t h e bank of l i g h t s above t h e c u l t u r e s and 3,000 lumens per f o o t 2 from t h e bank of l i g h t s below t h e c u l t u r e s .

The h o r i z o n t a l p o s i t i o n of t h e

b o t t l e s , which caused t h e c u l t u r e media t o have a s u r f a c e a r e a of 950 cm2 and m a x i m u m depth of the cultures.

7 cm, i n c r e a s e d t h e l i g h t f l u x i n t e r c e p t i o n of

We found t h a t such i n t e n s e i l l u m i n a t i o n s i g n i f i c a n t l y

increased purple membrane s y n t h e s i s i n t h e growing b a c t e r i a , i n agreement with previously r e p o r t e d r e s u l t s . 5 The temperature of t h e c u l t u r e media was maintained between 38' and 40'

by use of supplementary 200 w a t t incandescent lamps (Westinghouse

s o f t w h i t e ) which were placed at v a r i a b l e d i s t a n c e s from t h e s i d e s of t h e carboys and provided an a d d i t i o n a l average i l l u m i n a t i o n of 400 lumens per f o o t 2 f o r each carboy.

The c u l t u r e s were a e r a t e d by use of

l a r g e pore a i r d i f f u s e r s which caused a r a p i d churning movement i n t h e c u l t u r e media.

The a i r used t o supply t h e c u l t u r e s with oxygen was

f i r s t d i f f u s e d through d i s t i l l e d water i n o r d e r t o avoid excessive evaporation of t h e c u l t u r e media.

Foaming of t h e c u l t u r e s w a s c o n t r o l l e d

166

BECHER AND CASSIM

by t h e sparing use of antifoam s i l i c o n s p r a y .

During t h e f i r s t 36 hours

of growth, t h e c u l t u r e s were a e r a t e d a t a r a t e of 250 l i t e r s p e r hour. This a e r a t i o n rate s a t u r a t e d t h e

i n i t i a l 6 hours of growth.

4

l i t e r c u l t u r e s with oxygen f o r t h e

The oxygen concentration t h e n dropped

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r a p i d l y t o approximately 10 p e r cent of t h e s a t u r a t e d l e v e l .

After

36

hours, t h e a e r a t i o n r a t e was a d j u s t e d t o 160 l i t e r s p e r hour f o r t h e remainder of t h e growth p e r i o d which maintained t h e oxygen concentration

a t l e s s than 5 p e r cent s a t u r a t i o n .

mum purple membrane s y n t h e s i s began.

A t t h i s oxygen c o n c e n t r a t i o n , m a x i A f t e r 110 hours of growth, t h e

b a c t e r i a c u l t u r e s , which had been i n s t a t i o n a r y growth phase (absorbance

of 2 . 2 at 560 nm) f o r 30 hours, were harvested. P u r i f i c a t i o n of Purple Membrane.

The c e l l s from 4 l i t e r s of c u l t u r e

were p e l l e t e d by c e n t r i f u g a t i o n at 6 , 0 0 0 5 f o r 10 minutes ( S o r v a l l GSA rotor).

The r e s u l t i n g dark purple-red p e l l e t was then placed i n 2

l i t e r s of double d i s t i l l e d water with 5 mg mixed i n a blender.

of DNAase and immediately

The l y s a t e w a s then incubated i n a

with constant s t i r r i n g f o r

4 hours and c e n t r i f u g e d a t

minutes ( S o r v a l l SS-34 r o t o r ) ,

16' water bath

5 0 , O O O g f o r 30

The r e s u l t i n g s u p e r n a t e , which was

orange-red due t o bacterioruberin-containing membrane fragments, was removed l e a v i n g a purple p e l l e t .

The p e l l e t was then resuspended i n 800

m l of d o u b l e - d i s t i l l e d water and incubated a t 16' with constant s t i r r i n g .

f o r another

4

hours

The membrane suspension was t h e n c e n t r i f u g e d

a t 50,OOOg f o r 30 minutes t o f u r t h e r remove b a c t e r i o r u b e r i n membrane contaminants.

The l a t t e r process was r e p e a t e d , using 400 m l of double

d i s t i l l e d water, t h r e e o r f o u r times u n t i l no t r a c e of r e d contaminant could be seen i n t h e supernate a f t e r c e n t r i f u g a t i o n .

Centrifugation

of t h e purple membrane suspensions f o r 60 minutes a t 50,000gwas found t o be necessary i n t h i s l a s t s e t of p u r i f i c a t i o n procedures t o prevent excessive loss of purple membrane t o t h e supernate.

However, a l o s s

167

ISOLATING PURPLE MEMBRANE OF H. halobium of 2-5% of t h e purple membrane a f t e r c e n t r i f u g a t i o n was unavoidable s i n c e a d d i t i o n a l time o r g r a v i t a t i o n f o r c e during c e n t r i f u g a t i o n

re-

s u l t e d i n a tightly-packed purple p e l l e t which was d i f f i c u l t t o suspend i n water.

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The p u r p l e membrane w a s then f u r t h e r p u r i f i e d by use of a s t e p sucrose g r a d i e n t .

The purple membrane p e l l e t from t h e l a s t C e n t r i f u g a t i o n

w a s resuspended i n a minimum of double d i s t i l l e d water and l a y e r e d on

a s t e p sucrose gradient with increments of 45,40,38,36 and

(v/w).

16% sucrose

A f t e r c e n t r i f u g a t i o n f o r 8 hours a t 2 8 0 , 0 0 0 ~(Beckman SW-41 T I

r o t o r ) t h e purple l a y e r was s e p a r a t e d i n t o t h r e e p u r p l e bands of increasing p u r i t y as shown i n Figure l c .

A f a i n t r e d band was sometimes

s e e n d i r e c t l y above but c l e a r l y s e p a r a t e d from t h e purple band.

Frac-

t i o n 1 was found t o be t h e p u r e s t p r e p a r a t i o n , f r a c t i o n 2 w a s n e a r l y as p u r e , and f r a c t i o n 3 d i s t i n c t l y contaminated.

Generally, f r a c t i o n 3

DENSITY GRADIENTS-% SUCROSE

,

(WN)

16

-Red -F3

36

-F2

38

-F1

40 (1.5 M)

45

la

1b

-FIGURE

1c 1

Diagram of t h r e e sucrose density g r a d i e n t s employed i n purple membrane p u r i f i c a t i o n a f t e r c e n t r i f u g a t i o n at 280,OOOg f o r 8 hours. ( A ) Linear gradient from 16% t o 43% sucrose (w/w). ( B ) Linear g r a d i e n t from 36% t o 43% with a 1656 sucrose l a y e r above. ( C ) Step g r a d i e n t with increments of 45,40,38,36, and 16% s u c r o s e . The purple membrane p r e p a r a t i o n separated i n t o t h r e e bands of i n c r e a s i n g p u r i t y : F r a c t i o n 1 ( F l ) , Fraction 2 ( F 2 ) , and F r a c t i o n 3 (F3).

168

BECHER AND CASSIM

which contained approximately 20% of t h e p u r p l e membrane, was discarded. Fractions 1 and 2 , which contained equal amounts of t h e remaining purp l e membrane, were dialyzed a g a i n s t changes of double d i s t i l l e d water at

4' t o remove t h e sucrose.

F i n a l l y , t h e two f r a c t i o n s were c e n t r i -

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fuged a t 50,OOOg f o r 60 minutes t o remove any remaining r e d membrane contaminant and t h e r e s u l t i n g p u r p l e p e l l e t s were resuspended i n 0.Ol.M potassium phosphate b u f f e r at pH

7.

The f i n a l y i e l d of p u r i f i e d purple

membrane from 4 l i t e r s of Halobacterium halobium

w a s 50 m l of

purple membrane with absorbance of 2 . 0 a t 567 nm f o r f r a c t i o n 1 and f o r fraction 2.

Purple membrane p r e p a r a t i o n s were not p u r i f i e d i n t h e dark although precautions were taken t o avoid exposure of t h e p r e p a r a t i o n s t o i l l u m i n a t i o n i n excess of 50 lumens p e r foot' process.

throughout t h e p u r i f i c a t i o n

The p r e p a r a t i o n s were dark adapted f o r spectroscopic study by

s t o r i n g at 4'

i n t h e dark f o r 24 hours.

Light-exposure of purple

membrane p r e p a r a t i o n s was accomplished with an i l l u m i n a t i o n of 1,000 lumens per foot2 from two 40 watt f l u o r e s c e n t l i g h t s .

The p r e p a r a t i o n s

were maintained a t room temperature (approximately 23')

during t h e 15

minutes t h e preparations were light-exposed. Absorption Spectroscopy.made a t room temperature.

A l l spectrophotometric measurements were Purple membrane p r e p a r a t i o n s were d i l u t e d s o

t h a t t h e absorbance at 567 nm of light-exposed p r e p a r a t i o n s v a r i e d from 0.5 t o 1 . 0 .

Recordings of t h e absorption s p e c t r a were made a t t h e

scanning speed of 1 nm p e r second and a t t h e f u l l s c a l e absorption range of 1. O .

The d i l u t e caratenoid-containing supernate was recorded

a t a scanning speed of 1 nm p e r second and f u l l s c a l e absorption range of 0 . 2 .

The d e r i v a t i v e of t h e absorption s p e c t r a of t h e purple membrane

f r a c t i o n s w a s recorded with d i l u t i o n s such t h a t absorbance a t 567 nm of

169

ISOLATING PURPLE MEYBIBRANE OF H . halobium l i e h t - e x p o s e d p r e p a r a t i o n s v a r i e d from 1 . 0 t o 2 . 5 .

A s c a n n i n p s p e e d of

1 nm p e r second was m a i n t a i n e d and a f u l l s c a l e a b s o r p t i o n range of 0.2 t o 1 . 0 w a s employed t o p r o v i d e s u i t a b l e amplitude f o r a n a l y s i s . Recordings were made w i t h t h e sample and r e f e r e n c e c e l l s p l a c e d i n t h e

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sample h o l d e r of t h e s c a t t e r e d t r a n s m i s s i o n a c c e s s o r y w i t h t h e end-on p h o t o m u l t i p l i e r t u b e as c l o s e as p o s s i b l e t o t h e c e l l s .

S l i t widths

were m a i n t a i n e d from approximately 0.004 t o 0.04 nm o v e r t h e s p e c t r a l range u t i l i z e d t o maximize s i g n a l - t o - n o i s e not g r e a t e r t h a n

+_

ratio.

Wavelength e r r o r was

0.9 nm o v e r t h e s p e c t r a l range 300-800 nm and + -

0.1

nm over t h e 250-300 nm r a n g e .

Electron

Microscopy.

P u r p l e membrane p r e p a r a t i o n s were d i a l y z e d a g a i n s t

double d i s t i l l e d w a t e r and d i l u t e d u n t i l absorbance at 567 nm w a s 0.06. The p r e p a r a t i o n s were t h e n a p p l i e d d i r e c t l y t o carbon-coated

Formvar

g r i d s and were allowed t o dry at room t e m p e r a t u r e and h u m i d i t y .

Shadow-

ir.g was accomplished w i t h germanium (at 10 cm and a t an a n g l e o f 16 degrees).

E l e c t r o n micrographs were t a k e n w i t h a Zeiss EM 9s t r a n s -

m i t t i n g e l e c t r o n microscope. Disc G e l E l e c t r o p h o r e s i s .

Sodium dodecyl s u l f a t e polyacrylamide g e l

e l e c t r o p h o r e s i s was employed w i t h t h e p u r i f i e d p u r p l e membrane p r o t e i n a c c o r d i n g t o t h e s t a n d a r d p r o c e d u r e s d e s c r i b e d by Weber, P r i n g l e , and Osborn.

P u r i f i e d p u r p l e membrane w a s d i s s o c i a t e d w i t h sodium

dodecyl s u l f a t e (SDS) and l a y e r e d on 10% acrylamide g e l s 5 meter and 8 cm i n l e n g t h . p g were used f o r each g e l .

m i n dia-

C o n c e n t r a t i o n s o f p r o t e i n were approximated

from t h e p u r p l e membrane molar a b s o r p t i o n c o e f f i c i e n t of 6. 3 x 10 M-lcm-l

o r 60

P r o t e i n samples o f a p p r o x i m a t e l y 30 ug

3

a t 567 nm and t h e molecular weight o f 26,000 f o r t h e p u r p l e

~ l e c t r o p h o r e s i s was performed membrane p r o t e i n as p r e v i o u s l y r e p ~ r t e d . E with an i n i t i a l c u r r e n t of 3 m i l l i a m p e r e s p e r g e l u n t i l all t h e sample

BECHER AND CASSIM

170

entered t h e g e l (approximately 30 m i n u t e s ) .

A c u r r e n t of

8 milliamperes

was then employed f o r 4.5 hours with t h e t r a c k i n g dye migrating

6.5 cm.

The g e l s were s t a i n e d using Coomasie b r i l l i a n t b l u e a t room temperature f o r 10 hours.

A d e s t a i n e r and d e s t a i n i n g s o l u t i o n of 7.5% acetone and

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5% methanol was employed t o remove t h e s t a i n from t h e g e l s .

The

molecular weight of t h e polypeptide chain of t h e purple membrane p r o t e i n w a s determined from a l i n e a r p l o t of t h e logarithm of t h e molecular

weight versus mobility using t h e s t a n d a r d p r o t e i n s serum albumin, pepsinogen, t r y p s i n , myoglobin, and lysozyme.

The molecular weights

of t h e polypeptide chain of t h e s e s t a n d a r d p r o t e i n s a r e 68,000, 42,000, 23,300, 17,200, and 14,300 daltons r e s p e c t i v e l y . RESULTS Absorption Spectroscopy. Figure 2 i s an o r i g i n a l t r a c e of t h e v i s i b l e absorption s p e c t r a a s s o c i a t e d with purple membrane p u r i f i c a t i o n . A i s t h e spectrum

Trace

of t h e bacterioruberin-containing supernate a f t e r

c e n t r i f u g a t i o n of t h e b a c t e r i a c e l l l y s a t e .

Dominate f e a t u r e s of t h e

spectrum a r e maxima l o c a t e d at 470,498, and 533 nm.

Trace B i s a

t y p i c a l spectrum of light-exposed purple membrane from f r a c t i o n 3 of t h e sucrose g r a d i e n t immediately a f t e r d i a l y s i s of t h e f r a c t i o n a g a i n s t double d i s t i l l e d water.

The spectrum includes a maximum a t 563 nm and

d i s t i n c t shoulders a t 498 nm and 470 nm.

These shoulders a r e i n d i c a t i v e

of b a c t e r i o r u b e r i n contamination and correspond t o t h e contaminant maxima a t t h e same wavelengths as shown i n Trace A.

Trace C i s t h e

spectrum of t h e light-exposed of f r a c t i o n 1 of t h e sucrose g r a d i e n t a f t e r d i a l y s i s a g a i n s t double d i s t i l l e d water.

The main f e a t u r e of

t h e spectrum i s a m a x i m u m a t 567 nm due t o absorption of b a c t e r i o rhodopsin.

Comparison of Trace C with Trace B i n d i c a t e s a narrowing

of t h e band and a small red s h i f t of t h e maxima from 563 run t o 567 nm.

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ISOLATING PURPLE MEMBRANE OF H. halobium

I

350

I

450

1

550

171

I

650

I

750

WAVELENGTH [nm] FIGURE 2 O r i g i n a l t r a c e of t h e v i s i b l e absorption s p e c t r a a s s o c i a t e d with purple membrane p u r i f i c a t i o n procedures. ( A ) The s u p e r n a t e , containing (B) Fraction 3 b a c t e r i o r u b e r i n , from t h e l y s a t e of t h e b a c t e r i a c e l l . from t h e sucrose g r a d i e n t . (C) F r a c t i o n 1 from t h e sucrose g r a d i e n t .

These d i f f e r e n c e s can be i n t e r p r e t e d as t h e r e s u l t of a reduction of b a c t e r i o r u b e r i n contaminant.

However, although t h e shoulders i n Trace

C have been g r e a t l y reduced, they have not been completely e l i m i n a t e d

a t t h i s point i n t h e p u r i f i c a t i o n process.

BECHER AND CASSIM

172

Figure 3 i s an o r i g i n a l t r a c e of t h e absorption spectrum ( t r a c e A ) and t h e d e r i v a t i v e of t h e absorption spectrum ( t r a c e B ) of l i g h t exposed f r a c t i o n 1 i n phosphate b u f f e r a f t e r t h e f i n a l p u r i f i c a t i o n procedures.

The main f e a t u r e of t r a c e A i n t h e v i s i b l e spectrum i s an

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apparently s i n g l e band a t 567 nm due t o bacteriorhodopsin with no d e t e c t a b l e shoulders due t o b a c t e r i o r u b e r i n contamination.

This

r e s u l t should be compared with t h e t r a c e of t h e light-exposed f r a c t i o n 1 of t h e sucrose g r a d i e n t ( F i g . 2 , t r a c e C ) which had not y e t been

centrifuged t o remove any remaining b a c t e r i o r u b e r i n contaminants.

The

u l t r a v i o l e t spectrum of t r a c e A i n Figure 3 includes overlapping bands with extrema a t 290 nm, 280 nm and 274 nm.

The r a t i o of absorbance at

280 nm t o absorbance at 567 nm was 1 . 5 with,and 1 . 7 w i t h o u t , t h e use of t h e s c a t t e r e d transmission accessory of t h e spectrophotometer, i n d i c a t h g t h e n e c e s s i t y of minimizing l i g h t s c a t t e r i n g i n s p e c t r a l measurements.

This r a t i o was found t o be independent of membrane con-

c e n t r a t i o n s used i n t h i s study.

Our values f o r t h i s r a t i o should be

compared with t h e previously r e p o r t e d value of 2.0 3

.

The d e r i v a t i v e mode of p u r i f i e d f r a c t i o n 1 i n b u f f e r ( F i g . 3, t r a c e

B) i s f u r t h e r evidence of t h e absence of any bands i n t h e v i s i b l e spectrum of t h e purple membrane which could be a t t r i b u t e d t o contaminants such as b a c t e r i o r u b e r i n . The bacteriorhodopsin maximum at 567

nm i s

a l s o s u b s t a n t i a t e d by t h e d e r i v a t i v e spectrum.

The main f e a t u r e s of t h e absorption s p e c t r a of dark-adapted f r a c t i o n 1 i n b u f f e r a f t e r t h e f i n a l p u r i f i c a t i o n p-ocedures a r e s i m i l a r t o t h e light-perturbed p r e p a r a t i o n s .

However, t h e 567 nm

.

maximum i s b l u e s h i f t e d t o 559 nm and i s 12% l e s s i n t e n s e i n t h e darkadapted p r e p a r a t i o n s .

l e s s inThe p r o t e i n aromatic bands a r e only 1%

t e n s e i n t h e dark-adapted p r e p a r a t i o n s with no d e t e c t a b l e band s h i f t s .

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ISOLATING PURPLE MEMBRANE OF H. halobium

173

FIGURE 3 O r i g i n a l t r a c e of t h e absorption s p e c t r a (A) and d e r i v a t i v e of t h e absorpt i o n s p e c t r a (B) of p u r i f i e d purple membrane f r a c t i o n 1 i n 0.01 M potassium 350 nm) were reV i s i b l e wavelengths (800 nm phosphate b u f f e r (pH 7.0). corded with 50 nm p e r d i v i s i o n . U l t r a v i o l e t wavelengths (350 nm - 230 nm) were recorded with 20 nm per d i v i s i o n .

-

The spectroscopic e f f e c t s of l i g h t p e r t u r b a t i o n a r e completely r e v e r s i b l e .

It i s apparent t h a t our rigorous p u r i f i c a t i o n procedures have not measurably a f f e c t e d t h e previously observed l i g h t s e n s i t i v i t y f e a t u r e s of t h e p u r p l e membrane p r e p a r a t i o n s . Electron Microscopy.

The p u r i f i e d purple membrane p r e p a r a t i o n s from

f r a c t i o n s 1 and 2 c o n s i s t of round-to-oval

membrane s h e e t s with diameters

of 0 . 2 pm t o 0.6 pm as seen i n t h e e l e c t r o n micrograph ( F i g . 4 ) .

Purity

of t h e p r e p a r a t i o n i s demonstrated by t h e apparent l a c k of contaminating

BECHER AND CASSIM

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174

FIGURE.

4

Electron micrograph of purified p u r p l e membrane f r a c t i o n 1. (x 35,000)

ISOLATING PURPLE MEMBRANE OF H . halobium

175

membrane fragments a s s o c i a t e d with t h e purple membrane d i s k s .

This i s

i n c o n t r a s t t o f r a c t i o n 3 p r e p a r a t i o n s i n which contaminating membrane fragments were found a s s o c i a t e d with t h e purple membrane d i s k s . Disc Gel E l e c t r o p h o r e s i s . A l l t h e p r o t e i n from t h e p u r i f i e d p u r p l e

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membrane p r e p a r a t i o n from f r a c t i o n 1 migrates i n SDS acrylamide g e l as a s i n g l e band.

This r e s u l t provides a d d i t i o n a l evidence of t h e p u r i t y

of our purple membrane p r e p a r a t i o n and i n d i c a t e s t h e e x i s t e n c e of only one type of p r o t e i n i n purple membrane.

The molecular weight o f t h e

polypeptide chain of t h e purple membrane i s found t o be approximately 20,000 d a l t o n s .

This r e s u l t i s i n c o n t r a s t t o t h e previously r e p o r t e d

value of 26,000 daltons which was a l s o determined by SDS g e l electrophoresis.

3

DISCUSSION The p u r i f i c a t i o n methods described d i f f e r s i g n i f i c a n t l y from previously r e p o r t e d methods i n two important ways.

F i r s t , the recently

reported p u r i f i c a t i o n techniques f o r p u r p l e membrane include t h e r e suspension of c e n t r i f u g e d b a c t e r i a c e l l s i n a s o l u t i o n c o n t a i n i n g 25% sodium c h l o r i d e , and t h e d i a l y s i s of t h e suspension a g a i n s t d i s t i l l e d The purple membrane i s t h e n s e p a r a t e d from t h e b a c t e r i o r u b e r i n membrane by c e n t r i f u g a t i o n and washing of t h e purple p e l l e t with s a l t s o l u t i o n s and d i s t i l l e d water.

However, we have found t h a t r a p i d ex-

posure of h a l o p h i l i c b a c t e r i a c e l l s t o l a r g e volumes of water causes a l a r g e r degree of osmotic shock as previously reported.”

This method of

c e l l l y s i s , followed by repeated d i r e c t exposure of contaminated purple f r a c t i o n s t o l a r g e volumes of d i s t i l l e d w a t e r , y i e l d s p r e p a r a t i o n s of g r e a t e r p u r i t y than t h e d i a l y s i s method

as determined i n our l a b o r a t o r y .

A second d i f f e r e n c e between t h i s p u r i f i c a t i o n method and previous methods involves t h e use of a f i v e s t e p sucrose g r a d i e n t a s described i n t h e methods s e c t i o n .

O r i g i n a l l y , we had l a y e r e d t h e purple membrane on

BECHER AND CASSIM

176 a l i n e a r 0.5 M

-

1 . 5 M sucrose g r a d i e n t i n accordance with previously

reported p u r i f i c a t i o n techniques . 3 y 4

A purple band corresponding t o

sucrose concentrations from 36 t o 43% sucrose ( w / w ) was formed a f t e r c e n t r i f u g a t i o n f o r 8 hours a t 280,00og_ ( F i g . 1 A ) .

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then r e s t r u c t u r e d s o t h a t t h e

The g r a d i e n t was

36 t o 43% sucrose s e c t i o n i n t h e g r a d i e n t

was doubled i n length with a 16% sucrose l a y e r d i r e c t l y above ( F i g . 1 B ) . A f t e r c e n t r i f u g a t i o n , t h e upper s e c t i o n s of t h e r e s u l t i n g purple band was found by absorption spectroscopy t o be d i s t i n c t l y more contaminated than t h e lower s e c t i o n s .

The s t e p g r a d i e n t ( F i g . 1C) was then employed

t o s e p a r a t e t h e purple band of f i g u r e s

IA and 1 B i n t o t h r e e bands of

i n c r e a s i n g p u r i t y as described i n t h e methods s e c t i o n . The p u r i t y of t h e purple membrane p r e p a r a t i o n was analyzed using absorption spectroscopy, e l e c t r o n microscopy, and g e l e l e c t r o p h o r e s i s . Careful measurement of t h e v i s i b l e absorption s p e c t r a of t h e light-exposed p r e p a r a t i o n demonstrated p u r i t y by a band at

567 nm which was f r e e

of any shoulders at 498 nm and 470 nm due t o b a c t e r i o r u b e r i n contamination. Furthermore, t h e p r o t e i n t o chromophore r a t i o , as determined by aromatic amino a c i d absorption a t 280 nm and chromophore absorption a t

567 nm,

was found t o be 1 . 5 compared t o previously r e p o r t e d r a t i o values of 2.0.’

Larger values of t h i s r a t i o , vhich a r e i n d i c a t i v e of contaminating

p r o t e i n i n t h e purple membrane p r e p a r a t i o n , were found t o be accompanied by t h e appearance of t h e shoulders a t 498 nn and 470 nm i n t h e v i s i b l e spectrum.

Electron microscopy provided f l r t h e r evidence of p r e p a r a t i o n

p u r i t y as i n d i c a t e d by t h e apparent l a c k of contaminating membrane f r a g ments a s s o c i a t e d with t h e purple membrane i n e l e c t r o n micrographs.

Disc

g e l e l e c t r o p h o r e s i s of SDS d i s s o c i a t e d p r o t e i n from t h e p u r i f i e d purple membrane produced a s i n g l e band corresponding t o a polypeptide molecular weight of 20,000 d a l t o n s . fre e preparation.

This r e s u l t i s a l s o i n d i c a t i v e of a contaminant

ISOLATING PURPLE MEMBRANE OF H. halobium

177

Two f i n a l comments concerning t h e i s o l a t i o n o f p u r p l e membrane

from Halobacterium h a l o b i m are i n o r d e r .

F i r s t , s t u d i e s of t h e purple

membrane from t h e mutant Halobacterium halobium NRL R M which c o n t a i n s 11 only t r a c e amounts o f b a c t e r i o r u b e r i n i n v i v o have been r e p o r t e d . 4 9 1 1 As

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a r e s u l t t h e v i s i b l e s p e c t r a of t h e s e p u r p l e membrane p r e p a r a t i o n s do n o t d i s p l a y t h e contaminating b a c t e r i o r u b e r i n s h o u l d e r s a t

498 and

470 nm.

There i s , however, no evidence t h a t t h e loss of t h e b a c t e r i o r u b e r i n i n t h i s mutant would a l s o r e s u l t i n t h e loss of t h e c o n t a m i n a t i n g membrane with which t h e b a c t e r i o r u b e r i n chromophore i s normally a s s o c i a t e d .

In

f a c t , t h e u s e o f such a mutant a c t u a l l y e l i m i n a t e s a s e n s i t i v e i n d i c a t o r

( i . e . , t h e 498 nm and 470 nm s h o u l d e r s ) f o r d e t e c t i o n o f t h i s membrane contamination i n t h e p u r p l e membrane p r e p a r a t i o n s .

It i s noteworthy

t h a t p u r p l e membrane which was i s o l a t e d by t h e most r e c e n t l y r e p o r t e d t e c h n i q u e s from t h e c a r a t e n o i d producing s t r a i n

R1

Halobacterium h a l o b i m

%*asr e p o r t e d t o sometimes c o n t a i n small amounts of b a c t e r i o r u b e r i n .

4

Secondly, a b s o r p t i o n s p e c t r a of p u r p l e membrane p r e p a r a t i o n s have been p r e s e n t e d only s c h e m a t i c a l l y i n a l l b u t two of t h e p r e v i o u s s t u d i e s .

4,11

In t h e s e two s t u d i e s , as a l r e a d y d i s c u s s e d , t h e p u r p l e membrane p r e p a r a t i o n w a s i s o l a t e d from a s t r a i n of Halobacterium halobium which does n o t produce bacterioruberin.

A s a r e s u l t , c r i t i c a l p u r i t y d e t e r m i n a t i o n of t h e

p u r p l e membrane p r e p a r a t i o n s from p r e v i o u s l y r e p o r t e d a b s o r p t i o n s p e c t r a

is

not p o s s i b l e .

I n t h i s s t u d y c r i t i c a l s p e c t r a l methods, i n a d d i t i o n

t o e l e c t r o n microscopy and g e l e l e c t r o p h o r e s i s , were employed i n an e f f o r t t o e s t a b l i s h a c r i t e r i a of p u r i t y I n t h e d e s c r i b e d p u r p l e membrane prep-

-

a r a t i o n o f Halobacterium halobium R

1'

A p r e l i m i n a r y r e p o r t o f t h i s s t u d y w a s p r e s e n t e d at t h e B i o c h e m i s t r y / Biophysics 1974 Meeting, Minneapolis, Minnesota, June 2-7, P r o c . , Fed. h e r . SOC. Exp. B i o l .

2 (5)

1408).

1974 (Fed.

BECHER AND CMSIM

ACKNOWLEDWNTS The authors g r a t e f u l l y acknowledge t h e valuable advice and a s s i s t a n c e of Professor R .M. P f i s t e r , Department of Microbiology, The Ohio S t a t e University.

The authors thank D r . Walter S p e r l i n g , I n s t i t u t

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f c r Neurobiologie, J i i l i c h , Germany, f o r h i s generous g i f t of t h e mutant Halobacterium halobium R

1

used i n t h i s study.

This work was supported

i n p a r t by an Ohio S t a t e University g r a n t .

REFERENCES -

34, 365

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W. Stoeckenius and R . Rowen, J . C e l l B i o l . ,

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41,

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthe...
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