2,3-Dioxo-5-indolinesulfonic acid, a new highly specific reagent for modification of tryptophan in peptides and proteins.

A new reagent, 2,3-dioxo-5-indolinesulfonic acid (DISA), has been investigated for its specificity to react with tryptophan and tryptophan residues in peptides and proteins. On reaction of 19 amino acids with 1 M excess of DISA in 0.1 M acetic acid (pH 2.9), considerable (50%) modification of tryptophan was obtained within 50 min and no other amino acid was modified. After reaction for 5 h, only proline showed very slight (7%) modification. On reaction in the presence of 5 M excess of DISA, tryptophan was very rapidly modified. Modification of proline became appreciable in the presence of this molar excess of reagent and cysteine modification, although much smaller, became detectable (12 h, 7%). However, proline modification was completely prevented when this amino acid was engaged in a peptide linkage, even after reaction for 45 h in the presence of 5 or 10 M excess of DISA per proline residue. Reaction of egg albumin with 50 M excess of DISA was entirely specific for tryptophan and showed no modification of proline or cysteine residues. The reagent offers the advantages of stability, easy handling, high water solubility and high specificity. It affords protein and peptide derivatives that are completely water soluble because of the polar nature of the added group. The yellow color (lambda max, 367 nm) of the derivatives offers advantages of easy determination of the extent and location of the modification.

Resolution of D- and L-galactose peracetates as their bis(ethyl L-lactate) acetals by gas-liquid chromatography.

Reaction between ethyl L-lactate and each of a pair of sugar enantiomers, the peracetylated D-galactose and L-galactose diethyldithioacetals, produced two acyclic diasterioisomers. They could be separated by conventional gas-liquid chromatography. The corresponding fucose diastereomers were also separated. This process should make it possible to develop a general analytical method by which small amounts of enantiomeric sugars can be identified and their quantities measured.

Can an antibody-combining site be mimicked synthetically? The possible surface simulation synthesis of two antibody-combining sites complementary to two antigenic sites of lysozyme.

Our recent determination of the complete antigenic structure of native lysozyme was made possible by our introduction of the "surface simulation" synthetic concept. The remarkable success afforded by this strategy in the synthetic construction of antigenic sites prompted us to investigate whether it can be applied to mimic antibody-combining sites. Two peptides, designed to be complementary to antigenic sites 2 and 3 of lysozyme, were synthesized and their immunochemistry was studied. Each of the two complementary peptides exhibited an appreciable inhibitory activity towards the reaction of lysozyme with its antisera. Peptide immunoadsorbents bound only lysozyme and not antibody or myoglobin. Neither of the two peptides had any immunochemical activity in the myoglobin or bovine serum albumin immune systems. Furthermore, it was shown that three control synthetic peptides of myoglobin, of similar charge but different sequence, had no inhibitory effect on the lysozyme immune reaction. The evidence indicates that the antibody-combining sites sgainst antigenic sites 2 and 3 of native lysozyme were successfully mimicked synthetically, at least in terms of binding function.

Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid.

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.