HPLC Separation of Cholecystokinin Peptides-Two ...

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Dec 5, 2006 - ISSN: 0148-3919 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ljlc19. HPLC Separation of Cholecystokinin Peptides-Two.
Journal of Liquid Chromatography

ISSN: 0148-3919 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ljlc19

HPLC Separation of Cholecystokinin Peptides-Two Systems Margery C. Beinfeld , Robert T. Jensen & Michael J. Brownstein To cite this article: Margery C. Beinfeld , Robert T. Jensen & Michael J. Brownstein (1980) HPLC Separation of Cholecystokinin Peptides-Two Systems, Journal of Liquid Chromatography, 3:9, 1367-1371, DOI: 10.1080/01483918008062782 To link to this article: http://dx.doi.org/10.1080/01483918008062782

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JOURNAL OF LIQUID CHROMATOGRAPHY, 3(9), 1367-1371 (1980)

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WLC SEPARATION OF CHOLECYSTOKININ PEPTIDES--TWO

SYSTEMS

b Margery C. B e i n f e l d a s b , Robert T. Jensen', Mic ha e l J. Brow nste in aDepartment o f P s y c h i a t r y Washington U n i v e r s i t y Med i ca l School S t . L o u i s , M i s s o u r i 63110 b L ab o r at o r y of C l i n i c a l S c i e n c e N a t i o n a l I n s t i t u t e o f Mental H e a l t h B et h es d a, Maryland 20205 CSect i o n o n G a s t r o e n t e r o l o g y D i g e s t i v e Disease Branch N a t i o n a l I n s t i t u t e of A r t h r i t i s , Metabolism and D i g e s t i v e Diseases National I n s t i t u t e of Health B et h es d a, Maryland 20205 ABSTRACT

Two HPLC s y s t e m s are d e s c r i b e d which s e p a r a t e c h o l e c y s t o k i n i n (CCK) p e p t i d e s from e a c h o t h e r an d from g a s t r i n . One s y s t e m u t i l i z e s a S u p e l c o s i l LC-18 reverse p h as e column w i t h a n a c e t o n i t r i l e t r i e t h y l a m i n e p h o s p h a t e b u f f e r . The o t h e r syste m u t i l i z e s a V a ria n 2000 SW micropack r m l e c u l a r sieve column r u n i n 0.1 M p h o s p h a t e b u f f e r . These HPLC s y s t ems o f f e r improved r e s o l u t i o n o v e r conv e n t i o n a l g e l f i l t r a t i o n o f CCK and s h o u l d b e u s e f u l , when c ouple d w i t h radioimmunoassay, f o r d e t e r m i n i n g t h e m o l e c u l a r forms of CCK p r e s e n t i n b i o l o g i c a l samples. INTRODUCTION Several c h o l e c y s t o k i n - l i k e p e p t i d e s o c c u r i n v a r y i n g propor-

t i o n s i n d i f f e r e n t t i s s u e s (1).

Some o f t h e s e are undoubte dly t h e

b i o l o g i c a l l y a c t i v e , s e c r e t e d forms o f t h e hormone; o t h e r s are probably precursors o r i n a c t i v e metabolites. post-mortem a r t i f a c t s .

S t i l l o t h e r s may be

There h a s been c o n s i d e r a b l e e f f o r t made t o

d e v e l o p methods f o r s e p a r a t i n g and q u a n t i t a t i n g t h e v a r i o u s CCK related peptides.

Sephadex g e l chromatography combined w i t h sequence-

1367 Copyright Q 1980 by Marcel Dekker. Inc.

1368

BEINFELD, JENSEX, ANTI BROWNSTEIN

s p e c i f i c r a d i o i m u n o a s s a y h a s b een u s ed f o r t h i s p u r p o s e , b u t t h i s c o m b i n a t i o n is n o t i d e a l .

The HPLC s y s t e m s d e s c r i b e d h e r e are a n

improvement i n s p e ed and r e s o l u t i o n o v e r c o n v e n t i o n a l g e l chromato g r a p h y sy s t e m s. MATERIALS S u l f a t e d and d e s u l f a t e d CCK8 were t h e g i f t o f D r . Miguel

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' O n d e t t i o f Squibb. P e n i n s u l a Labs.

Sigma.

D e s u l f a t e d CCK8 w a s also purc ha se d from

was purc ha se d from

CCK4 (Trp-Met-Asp-Phe-NH2)

CCK4, CCK5 (Gly-CCK4), CCK6 (Met-Gly-CCK4).

Met-Gly-CCK4). Jensen.

CCK7 (Tyr-

G a s t r i n 17 s u l f a t e d were t h e g i f t of D r . Robe rt

Gastrin 1 7 d e s u l f a t e d w a s p u r ch as ed from Calbiochem.

and CCK39 were t h e g i f t o f V l k t o r Mutt.

The f r e s h l y d i s t i l l e d t r i -

e t h y l a m i n e was t h e g i f t o f Tom O'Donohue. F i s h e r (HPLC g r a d e ) .

CCK33

A c e t o n i t r i l e was from

A Varian 5020 l i q u i d chromatograph e quippe d

w i t h a V e r i c h r i n d e t e c t o r was u s e d f o r t h e s e s t u d i e s .

The a b s o r b -

ance o f t h e samples w a s f o l l o wed at 210 nM. NETHODS A.

C - 1 8 Reverse Phase Systam A S u p e l c o s i l ( S u p e l c o ) column was e l u t e d w i t h a 22% a c e t o n i t r i l e -

78% p h o s p h o r i c a c i d - t r i e t h y l a m i n e b u f f e r , pH 6 . 5 ( 1 7 ml o f 85%

p h o s p h o r i c a c i d w a s added t o d i s t i l l e d deionized-Lblillipore

filtered

water and f r e s h l y d i s t i l l e d t r i e t h y l a m i n e is added u n t i l t h e pH r e a c h e s 6.5.

The volume o f t h e s o l u t i o n i s the n a d j u s t e d t o 1 1 ) .

The b u f f e r i s p a s s e d t h r o u g h a Waters C-18 s e p pack p r i o r t o u s e . B.

M o l e c u l a r S i e v e System A Varian 2000 SW micro pack m o l e c u l a r s i e v e column i s run w i t h

0 . 1 M Na p h o sp h a t e b u f f e r pH 7.0. s e p pack

p r i o r t o u s e.

The b u f f e r i s p a s s e d through a

1369

CHOLECYSTOKININ PEPTIDES C.

Sample P r e p a r a t i o n

The s t a n d a r d s are d i s s o l v e d i n a c o m p a t i b l e s o l v e n t (one t h a t d o e s n o t a b s o r b h e a v i l y a t 210 nM). p u r i f i e d on a s e p pack clogging.

B i o l o g i c a l s a m p l e s were p r e -

o r e x t e n s i v e l y e x t r a c t e d t o p r e v e n t column

A method which y i e l d s 55-60;; r e c o v e r y o f CCK-like material

from b i o l o g i c a l i s t o prewash s e p pack w i t h 15 ml a b s o l u t e methanol f o l l o w e d by 20 m l d i s t i l l e d water.

The sample i s a p p l i e d i n a n y

c o n v e n i e n t volume, washed w i t h 20 m l d i s t i l l e d water and e r u t e d

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w i t h 3 m l a b s o l u t e methanol.

The methanol i s e v a p o r a t e d and t h e

sample is r e d i s s o l v e d i n water and i n j e c t e d . s a m p l e s c a r e s h o u l d be t a k e n t o wash systems thoroughly

In separating biological

t h e columns and i n j e c t i o n

between i n j e c t i o n s of s t a n d a r d s and b i o l o g i c a l

materials so t h a t t h e b i o l o g i c a l materials a r e n o t c o n t a m i n a t e d by the standards.

It i s a d v i s a b l e i n o r d e r t o t e s t t h e t h o r o u g h n e s s

o f working p r o c e d u r e s , t o r u n a water b l a n k between s t a n d a r a s and b i o l o g i c a l samples and a s s a y t h e f r a c t i o n s i n t h e radioimmunocssay

.

S u b s t a n t i a l amounts o f p a r t i a l l y and c o m p l e t e l y o x i d i z e L CCR

are p r e s e n t i n some t i s s u e extracts.

Therefore, it i s importmt t o

know t h e r e t e n t i o n times of p a r t i a l l y and c o m p l e t e l y o x i d i z e d p e p t i d e s on t h e C-18 system.

To d e t e r m i n e t h e r e t e n t i o n t i m q i :

p a r t i a l l v and c o m p l e t e l y o x i d i z e d CCK, 1 u l of 30% hydrogen p e r o x i d e w a s added t o 50 ul of p e p t i d e (1 ug/ul) and a l i q u o t - . o f t h e r e s u l t i n g s o l u t i o n were chromatographed a t several t i m e s afterwards. RESULTS T a b l e 1 l i s t s t h e r e t e n t i o n times o f some CCK p e p t i d e s o C - 1 8 r e v e r s e phase column.

ch5

The peak w i d t h o f t h e p e p t i d e p e a x s are

s u f f i c i e n t l y narrow t h a t t h e r e i s b a s e l i n e s e p a r a t i o n betweep t h e peaks.

It i s a l s o p o s s i b l e t o s e p a r a t e t h e s u l f a t e d and d e s u l f a t e d

forms o f CCBand s u l f a t e d and d e s u l f a t e d g a s t r i n 1 7 .

Retention

times are r e p o r t e d f o r r e d u c e d , p a r t i a l l y and c o m p l e t e l y o x i d i z e d

BEINFELD, JENSEN, AND BROWNSTEIN

1370

TABLE 1 R e t e n t i o n Times o f CCK P e p t i d e s Run i n 222 AN-TFAP on S u p e l c o s i l LC-18 Column

Re t e n t ion Time

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Pep t i d e

8.9 3.0 8.9

cCK4 CCK4 o x i d i z e d CCKS G a s t r i n 17 s u l f a t e d G a s t r i n 17 d e s u l f a t e d Gastrin 17 d e s u l f a t e d o x i d i z e d CCK8 s u l f a t e d CCKB s u l f a t e d p a r t i a l l y o x i d i z e d CCKB s u l f a t e d c o m p l e t e l y o x i d i z e d CCKB d e s u l f a t e d CCX8 d e s u l f a t e d p a r t i a l l y o x i d i z e d CCKB d e s u l f a t e d c o m p l e t e l y o x i d i z e d CCK6

forms o f CCK.

11.7 16.5 5.5 17.4 5.1; 7 . 9 3.3 19.8 6.6, 9.8 3.7 21.2

CCK8 h a s two m e t h i o n i n e r e s i d u e s s o t h a t two p a r t i d -

l y o x i d i z e d forms are g e n e r a t e d w i t h two u nique r e t e n t i o n t i m e s . A f t e r several h o u r s a t 4'C

b o t h met h i o n i n e r e s i d u e s a r e o x i d i z e d

and a s i n g l e u n i q ue r e t e n t i o n t i m e is o b s e r v e d .

CCK4 and g a s t r i n

1 7 o n l y have one met h i o n i n e r e s i d u e so no p a r t i a l l y o x i d i z e d f o r m are g e n e r a t e d .

TABLE 2

R e t e n t i o n Times o f CCK P e p t i d e s Run i n 0 . 1 M Phospha te B u f f e r pH 7.0 on Varian 2000 SW Wicropack H o l e c u l a r S i e v e Colunm P ep t l d e

Gastrin 1 7 s u l f a t e d Caerulein ccK33 G a s t r i n 17 d e s u l f a t e d CCKB s u l f a t e d CCKB d e s u l f a t e d ca4

ccK.5

CCK6 CCK7 d e s u l f a t e d

Retent ion Time (minutes) 1 5 .O 15.1 15.2 16.3 19.8 23.4 25.6 25.9 27.8 31.3

CHOLECYSTOKININ PEPTIDES

1371

T a b l e 2 lists t h e r e t e n t i o n t i m e s of t h e CCK p e p t i d e s on t h e

molecular sieve column.

The column w a s d e s i g n e d t o s e p a r a t e mole-

c u l e s on t h e b a s i s o f molecular wei g h t b u t some p e p t i d e s run anomalously.

The column e a s i l y s e p a r a t e s some of t h e m a jor forms o f

CCK.

DISCUSSION

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Two HPLC s y s t e m s are d e s c r i b e d h e r e which p e r m i t r a p i d and complete s e p a r a t i o n o f

CCK p e p t i d e s .

U t i l i z i n g t h e s e systems

i n c o n j u n c t i o n w i t h CCK radioimmunoassay h a s f a c i l i t a t e d t h e t a s k o f d e t e r m i n i n g what molecular forms o f CCK are found i n b i o l o g i c a l

samples and how t h e y are i n t e r c o n v e r t e d . REFERENCES

1.

R e h f e l d , J.F., Immunochemical s t u d i e s o n c h o l e c y s t o i c i n i n II. D i s t r i b u t i o n and m o l e c u l a r h e t e r o g e n e i t y i n t h e c e n t r a l ne rvous s y s t e m and small i n t e s t i n e of man-and hog. 3. Biol. Chem. 253: 4022, 1978.