A method for the quantitative analysis of streptothricin hydrolysates

WOTIX

214 A method

’

for

the quantitative’

analysis of streptothricin

hydrolysates

As is well known, streptothricin antibiotics consist of two unusual basic amino acid residues, r+lysin and streptolydine, and one amino sugar residue, a-D-gulosamine. I-Iowever, the proportion of these compounds in the various types of streptothricins is little known. The only available data in the literature are for the antibiotics streptothricin, streptoline and racemomycin 0, whose structures are considered to have been established. In the course of a systematic study of streptothricins we met wi,th the necessity of developing a reliable method for the quantitative determination of the aforementioned constituents in the hydrolysates of these antibiotics. Since all the acid hydrolysis products of the! streptathricins (in&ding ammonia and x,6anhydro-/3-D-gulosamine, formed from a-rJ-gulosamine in the course of the hydrolysis) give the ninhydrin reaction, we used for our purpose the method of SPACKMAN, STELN AND MOOR&, modified by KOMINZ; so as to make it suitable for the analysis of sufficiently complex mixtures of ninhydrin-positive base@. After a number of preliminary experiments the following optimal conditions were established : column : 0.9 x 60 cm; resin : Arnberlite CG 120, fraction I3 (Evans Electroselenium Ltd., England) ; buffer: 0.70 N Na citrate, pII 5.28 & 9.02 ; temperature: 50”; rate: 30 ml/h; duration of run: 61/3 h (inlet of ninhydrin begun 1.5 h after start of run) ; apparatus : automatic amino acid analyzer type goIg/III (CzechoSlovakia). . In Fig. I, one curve depicts the results of analysis of a model mixture containing a collection of basic and aromatic amino acids and the constituents of the hydrolysate of one of the strtiptothricins (phytobacteriomycin D), It must be stressed that the 2.0 -

1.0; 0.8 -

0.4 -

PHENYLA$WNE TYROSINE~$~os;;: ~. ;A INE :: :::i :: :: :: :: ::::

LYS,NE :: :: :. AMMONIA

i : ts 9

STREPTOLYDINE h 60

90

120

150

180

210 Effluent

240 (ml)

Fig. I. Chromstographic analysis of a hydrolysatc of phytobsctoriomycin D. Dotted lines show the position of pealrs of the aromatic amino acicls, glucosamine and the common basic amino acicls.

resolving power, particularly with respect to streptolydine and ammonia, greatly depends on the pH of the effluent buffer. As one can see in Fig, I a-D-gulosamine, appearing immediately after glucosamine, occupies a position characteristic for another hexosamine, namely galactosamine 3. Table I gives the volumes of the buffer solution required for the elution of each substance and also the I-E x T/Vconstants of the substance, which after correction with reference to the value for one of the J.Ckronzatog.,

19 (x965) 2x4-215

NQTES

TABLE

2=5 I

STANDARD XN nd FOR

Colq!Wlzd

u X w CONSTANTS FOR CZECHOSLOVAKAMINO ACID ANALYZER L-@-LYs~N~, STREFTOLYDINE AND BASIC ~WNO ACIDS

-

Tyrosinc” Phenylalanine~ D-Glucosamine” D-Gulosamineo Lysinea x,4-Anhydrogulosaminee r.++Lysinee I-IisLidincn Ammonia SLrcptolydinee Argininefi

Effdzccnt

Slandard

volzlme W)

w x w conslast

i$ 70 33 =o3 107 ‘27 128 =65 179 242

AND

EFFPLUIZNT

VOLUME

x5.ro r5.73 x5.8x r7.53 X0.15 x6.02 r5.55 640 *5*9e

a The basic and aromatic amino acids were obtained from the California CorporaUon for Biochemical Research, Los Angeles 63, Calif., U.S.A. b D-Glucosaminc was obtained from Lhe Serva En~wicl~lungslabor, Neidelberg, Germany. 0 The samples of these compounds were isolated from the acid hydrolysalze of a phflobactcriomycin preparatian by partition chromatography on powdered celluIosc,

basic amino acids given in the table may be utilized for work with other types of instruments. This method may And convenient use in the analysis of many antibiotics of the strcptothricin series.

I D. N. SPACICMAN,W. II. STEIN AND S. MOORE, Anal. Chcm., 30 (rg58) ergo. 2 D. R. KOMINZ, J. C~~rorna~o~*, g (x962) 253. 3 I%. I?. WhLnono, B. F, COBB, M. ADAMS-MAYNE AND D. N. WARD, Anal. BiocJwn.,

6 (zg63)

367.

Received December rfith, xg64 J_ Cltromatog.,

A simple swrface

rapid eechnique

studying the

19 (1gG5) 2.x4-2r5

reaceians of

by paper

The screening large numbers bacterial isolates any one enzymic reaction usually a and laborious although it one which to be in investigations the changes floral activity soil, spoiling fish etc. following technique been developed that originally J. Claromalog.,

Ig(xg65)215-ZIG