Dinitrophenylation and Thiolysis as a Tool in Protein Chemistry

Abstract

The thiolytic cleavage of 2,4 dinitrophenyl derivatives of sulfhydryls, imidazole imino-nitrogens, and phenolic hydroxyls converts dinitrophenylation into a reversible method for masking or labeling these functional groups. The merits of this method are illustrated in the synthesis of peptides and polyamino acids (including a potent synthetic immunogen), in sharpening the specificity of dinitrophenylation, in masking "super reactive" nucleophiles in proteins while labeling "buried" functional groups, and in the probing and three-dimensional mapping of enzyme active sites. Analytical techniques for monitoring the rate and extent of the thiolysis step are described.Dinitrophenylation has played a major role in protein chemistry, as a key reaction used in the identification of N-terminal amino acids and thus in the elucidation of amino acid sequences of proteins [1]. Being an activated aryl halide, FDNB* reacts not only with the N-terminal amino groups but also with sulfhydryls, imino-groups of imidazoles, phenolic hydroxyls and e-amines [2][3][4]. Attempts have been made to achieve group specificity in dinitrophenylation by using less reactive reagents (e.g, dinitrobenzenesulfonic acid [5]) or by performing the reaction at a pH at which some of the functional groups are relatively much less reactive [6]. However, in practice it was found that the reactivity of certain functional groups in a protein is so much influenced by their microenvironment that it is often possible to find reaction conditions under which FDNB can be led quite selectively not only to one type of functional group but rather to a unique group within a specific site of the protein [7][8][9][10][11].A few years ago we showed [12] that DNP derivatives of sulfhydryls, imidazole irnino-nitrogens, and phenolic hydroxyIs (but not of Q and e amino groups) can be easily displaced by thiols, thus converting dinitrophenylation into a reversible tech-

Evidence for an interprotomerial active site in D‐glyceraldehyde‐3‐phosphate dehydrogenase

“…The fact that a considerable amount of the protein was dissociated into monomers is in accordance with our finding ([5] and unpublished results) that the yield of the two Cys-Lys crosslinked peptides which were isolated is relatively low (~20% in the peptic digest mixture), that other crosslinks which were identified are intraprotomerial [6] ( fig.1) and that the bifunctional reagent, once anchored to Cys-149, may undergo hydrolysis of its second C-F bond and thus end up as monovalent label.…”

“…Proposed structure for the active site (shaded area) of apo-GAPD. Arrows 1 and 2 indicate the crosslinks established by labeling with F, DNB [5,6], which suggest proximity of the three indicated functional groups in the three dimensional structure of the apo-enzyme.…”

“…Note that the Cys-149 and Lys-183 residues are suggested to be provided by two adjacent protomers (a and a') while Cys-149 and Cys-153 which are also crosslinked by F, DNB [6] are provided by the same protomer. the very anchoring of the bifunctional label onto Cys-149 may induce a conformational change bringing the -SH and -NH2 groups closer together; c) the structure of the protein in solution may differ from that of the crystalline enzyme.…”

“…One type of crosslink was formed between the sulfhydryl group of Cys-149 and the e-amino group of Lys-183 [5]. Another type of crosslink connected Cys-149 and Cys-153 [6] . It was shown that the crosslinks were formed within the tetrameric molecule, as there was no change in its sedimentation coefficient as a result of the labeling.…”

Modification of amino acid residues in the S 1 ′ binding site of carboxypeptidase Y

“…Tyrosine and histidine can be regenerated from O-DNP-tyrosine and N~%DNP-histidine by treatment with thiol compounds without affecting N'-DNP-lysine and N~ acids (19). Thus, provided a tyrosyl residue is the site of reaction with t4C-FDNB, treatment with mercaptoethanol should release the radioactive label attached to the enzyme.…”