ONE-DIMENSIONAL OF
DNS-AMINO Ya.
I.
CHROMATOGRAPHY
ACIDS UDC 577.156.41
Lapuk
E d m a n ' s method in combination with dansylation is frequently employed f o r d e t e r m i n i n g the a m i n o acid sequence of peptides, using two-dimensional c h r o m a t o g r a p h y to identify the dansyl[DNS]-amtno acids
[1]. However, not infrequently the o n e - d i m e n s i o n a l method of s e p a r a t i o n in s e v e r a l solvent s y s t e m s p r o v e s to be m o r e r e l i a b l e . H e r e we p r o p o s e a s c h e m e f o r the s u c c e s s i v e use of five s y s t e m s of organic solvents f o r the s e p a r a tion of 17 DNS-amino acids: 1) c h l o r o f o r m - t e r t - a m y l a l c o h o l - a c e t i c acid ( 7 0 : 3 0 : 0 . 5 ) [2]; 2) c h l o r o f o r m - e t h a n o l - a c e t i c acid (45 : 4 : 0.75) ; 3) a c e t o n e - i s o p r o p a n o l - c o n c e n t r a t e d a m m o n i a (44 : 36 : 5.6) [3]; 4) c h l o r o f o r m - e t h a n o l - a c e t i c acid ( 3 8 : 4 : 3 ) [4]; and 5) c h l o r o f o r m - i s o a m y l a l c o h o l - a c e t i c acid ( 4 5 : 5 : 2 . 5 ) . S y s t e m s 2 and 5 have not been d e s c r i b e d b e f o r e . C h r o m a t o g r a p h y was p e r f o r m e d on plates with d i m e n s i o n s of 10 × 10 c m with KSK-3 silica gel in a fixed l a y e r (10 g of silica gel, 1 g of g y p s u m , 70 ml of w a t e r , 22 p l a t e s , Stahl a p p a r a t u s f o r depositing the sorbent). Of the organic solvents, only the c h l o r o f o r m was additionally purified, and then 1.5% of ethanol was added to it. C h r o m a t o g r a p h y was p e r f o r m e d with the f r e s h l y p r e p a r e d solvent s y s t e m s (this applies p a r t i c u l a r l y to s y s t e m s 1 and 4). The plates w e r e dried in a c u r r e n t of hot a i r f o r 10-15 rain. A f t e r c h r o m a t o g r a p h y in s y s t e m s 1 and 2, the hydrophllic amino acids, the dic a r b o x y l i c acids, and the d a n s y l - s u l f o n i c acids r e m a i n e d at the s t a r t . All the hydrophobic amino acids w e r e s e p a r a t e d with the exception of alanitm and t y r o s i n e . If t h e r e was a spot c o r r e s p o n d i n g to alanine o r t y r o sine, it was c h r o m a t o g r a p h e d s e p a r a t e l y in s y s t e m 5. A f t e r s y s t e m s 1 and 2, c h r o m a t o g r a p h y was p e r f o r m e d in s y s t e m 3, in which all the hydrophobic amino acids m i g r a t e d upwards leaving a position f r e e f o r the s e p a r a t i o n of the hydrophilic amino acids - s e r i n e and threonine - and the dicarboxylic acids r e m a i n e d at the s t a r t . Subsequent c h r o m a t o g r a p h y in s y s t e m 4 led to the s e p a r a t i o n of a s p a r t t c and glutamic acids. TABLE 1
DNS-amino acids
Damylamide Isoleucine Valinc Leucine Proline Phenylalanine Alanine TGTmsine Yptophan Ty eine Dansylsuffonic acid Threortine Serine Lysine Histidine
. _ ~ n solvent systems 1,1314 5 0,911 -- I -o.721
-
0,661 -0,591 °:47 I °'54 0,43 I 0,36 [
o
-
-
-
-
-
-
I -
I -I -I -[ I -
-
-
-
0~0 0,26 0,3,t
Io,541 Z
0 10,441 o IO,381 0,431 I -
-
-
-
-
-
-
-
0,22
oO.,61 : i =
o,,o
°'361-
05_9
I -
All-Union S c i e n t i f i c - R e s e a r c h Institute of Genetics and the Breeding of Industrial M i c r o o r g a n i s m s . T r a n s l a t e d f r o m Khimiya P r i r o d n y k h Soedinenii, No. 1, pp. 117-118, J a n u a r y - F e b r u a r y , 1974. Original a r t i c l e submitted June 5, 1973.
© 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. No part o f this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission of the publisher. A copy o f this article is available from the publisher for $15.00.
134
So f a r as concerns the basic amino acids and methionine, on chromatography in systems 1 and 2 the arginine remained at the start, the Rf value of histldine coincided with that of glycine, that of lysine with phenylalanine, and that of methionine with alanine. These pairs were separated on additional chromatography in system 5. The results of the chromatographic separation of the DNS-amino acids in the five solvent systems are given in Table 1. LITERATURE 1. 2. 3. 4.
CITED
I. Rosmus and Z. Deyl, Chromatog. Rev., 13, 663 (1971). D. Morse and B. L. Horecker, Anal. Biochem., 14, 429 (1966). B. G. Belen'kii, ]~. S. Gankina, and V. V. Nester.v, Dokl. Akad. Nauk SSSR, 172, 92 (1967). Z. Deyl and I. Bosmus, J. Chromatog., 20, 514 (1965).
135
