Resolution of DL-Tryptophan by Affinity Chromatography on Bovine-Serum Albumin-Agarose Columns

Abstract

Bovine-serum albumin, known to have antipodal specificity in the binding of tryptophan, was selected as the affinity chromatographic matrix for the attempted chromatographic resolution of DL-tryptophan. Complete resolution was accomplished when DL-tryptophan was chromatographed on bovine-serum albuminsuccinoylaminoethyl-Sepharose.Resolution of optical isomers by conventional methods is frequently laborious and incomplete. The mechanical and chemical methods are empirical in nature, and in practice choosing the appropriate resolving agents and physical means of separation can be difficult. Resolutions based on highly specific biological properties have been limited to enzyme reactions, and these reactions always resulted in chemical modification of one of the isomers. Differential binding of some optical isomers by a few serum proteins has been reported. Examples include binding of DI-tryptophan to bovine-serum albumin (1) and dl-aldosterone to corticosteroid-binding globulin (2). It would seem that this property of steroselective binding of optical isomers by proteins could be used for resolution of some optical isomers. We have begun studies to investigate this possibility and report here the resolution of DL-tryptophan on affinity columns of bovine-serum albumin-succinoylaminoethyl-agarose. (5), in which dipeptides are formed by reaction of the tryptophan with -glutamic acid N-carboxyanhydride, the -glutamyl-D-tryptophan is separated from the L-glutamyl-L-tryptophan by ion-exchange chromatography, and each diastereomer is quantitated. LTryptophan content was determined by the method of Guilbault and Hieserman (6) with -amino-acid oxidase, and an Aminco fluoromicrophotometer with a CS 7-60 primary filter and a 47B, 2A combination secondary filter.The substituted agarose supports were prepared according to Cuatrecasas (7) with cyanogen bromide. The extent of coupling was determined from amino-acid analyses of hydrolysates of the support material, and coupling efficiencies of 5-6% were normally obtained. Chromatographic columns were packed with either bovine-serum albumin linked directly to the agarose beads (bovine-serum albumin-agarose), bovineserum albumin linked to the agarose beads by an ethylenediamine-succinic acid leash (bovine-serum albumin-succinoylaminoethyl-agarose), or defatted bovine-serum albumin linked to the agarose beads by the same leash (defatted bovine-serum albumin-succinoylaminoethyl-agarose). Control columns were packed with agarose or leashed agarose (no bovine-serum albumin) as appropriate. RESULTSPreliminary experiments in which bovine-serum albuminagarose columns were used for chromatography of DLtryptophan indicated that there was some resolution of the DI,-tryptophan on these columns. However, these columns did not give reproducible results, due to the fact that the column material seemed to be altered during regeneration. In contrast, chromatography of DL-tryptophan on either of the two leashed column materials was reproducible, and hence these column materials were used in...

Effects of a dietary excess of leucine on the metabolism of tryptophan in the rat: a mechanism for the pellagragenic action of leucine

“…However, [benzene ring U-14C]tryptophan was available only as the racemic mixture. This was resolved by exploiting the stereo-specific binding of tryptophan to serum albumin, by the following modification of the method of Stewart & Doherty (1973), using immobilized bovine serum albumin. Cyanogen bromideactivated CH Sepharose 4B (Pharmacia Ltd) was swollen in 1 mM-hydrochloric acid and was then washed on a sintered-glass disk with 1 mM-HC1.…”

The metabolism of l-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism

“…Studies with cell-free extracts have shown that, in addition to enzymes required for the kynurenine pathway, the liver possesses the necessary enzymic complement for the catabolism of tryptophan by each of the other three pathways (Weissbach et al, 1959;Civen & Knox, 1959;Renson et al, 1966 Radiolabelled DL-tryptophan was resolved optically, and L-l5-3Hltryptophan repurified, by affinity chromatography on bovine serum albuminSepharose 4B as described by Stewart & Doherty (1973). The sources of all other materials are detailed in Smith & Pogson (1980).…”

Drug binding to human serum albumin: Abridged review of results obtained with high‐performance liquid chromatography and circular dichroism

“…The application of immobilised serum proteins as chiral selectors stems from the seminal 1973 work of Stewart and Doherty, 27 who were able to resolve the two antipodes of tryptophan on a bovine-serum albumin (BSA)-sepharose column, in agreement with the stereoselectivity previously observed for the in vitro binding of tryptophan to BSA. Later, other groups 28,29 used BSA-and HSA-based sepharose columns to separate drug enantiomers (such as warfarin and benzodiazepines) and to determine binding constants for a variety of ligands, including fatty acids, steroids, and drugs.…”