Competitive labelling, a method for determining the reactivity of individual groups in proteins. The amino groups of porcine elastase

Abstract

1. A method is described for determining the ionization constants and reactivities of individual amino groups in proteins. The principle is that in the presence of a trace amount of radioactive label, the various reactive groups in a protein molecule will compete for the label and the amount incorporated into any one group will be determined by its nucleophilicity, pK and micro-environment. The relative amounts of label incorporated into various groups will be proportional to their second-order rate constants and by comparing these rate constants with those expected on the basis of a linear free-energy relationship obtained with a series of standard compounds, the micro-environment can be defined for a particular amino group. 2. The method consists of treating a protein and an internal standard with a limiting amount of radioactive reagent and then with an excess of unlabelled reagent to yield a chemically homogeneous but heterogeneously labelled compound. After appropriate enzymic digestion peptides containing each labelled group are isolated and their rates of reaction, relative to the internal standard, are determined from their specific radioactivities. The entire procedure is repeated at several pH values. 3. When the method was applied to the amino groups of porcine elastase by using tritiated acetic anhydride as the labelling reagent, the N-terminus was found to have pK(a) 9.7 and a much lower than normal reactivity. Lysine-87 and lysine-224 were found to have pK(a) 10.3 and normal reactivities. At pH values greater than 10.5 there are discontinuities in all the titration curves, indicating that the entire molecule is undergoing a structural reorganization.

Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping.

“…Furthermore, HEL yields complete sequence data by reproducibly forming abundant proteolytic peptide ions and has a highly stable tertiary structure. Thus, it appears to be well suited as a model protein in chemical modification studies that may affect tertiary structures and reactivities (6,9).…”

“…Chemical reactivities of amino acid residues have been mainly determined by competitive labeling techniques, which, however, are laborious and generally require radioactive probes (6). For example, acetylation of amino groups has been extensively used for modifying enzymatic properties, immunological reactivity, and proteolytic digestion patterns (7).…”

“…Incomplete acylation of proteins has been frequently observed (8), while free accessibility of reactive residues is generally anticipated (7). In spite of some success of this view [e.g., in differential labeling studies of free and antibody-bound antigens (9)], parameters like electrostatic interactions and other microenvironmental effects may obscure a direct understanding of amino acid reactivities (6). Thus, in addition to an accurate description of protein structure and dynamics, experimental approaches that rapidly provide detailed chemical reactivity data are needed in structure-function studies of proteins.…”

Ferredoxin binding site on ferredoxin: NADP+ reductase

“…a conformational change induced by chemical modification. To overcome this problem, the overall degree of labelling is best kept so low that the number of multiply modified protein molecules is negligible (Kaplan et al, 1971 ;Bosshard, 1979). In the case of the biotin-avidin system, probably for the first time used here to map a binding site by the differential protection technique, the degree of biotinylation had to be greater than one molecule biotidprotein molecule because the amount of FNR available in the experiment was limited.…”

Reactivity and Ionization Constants of the Lysine Residues in Apovitellenin I of Emu Egg Yolk Low-density Lipoprotein by Competitive Labelling

“…Kaplan and his associates (Kaplan et al 1971) have shown that the method of competitive labelling with electrophilic reagents in the presence of a standard nucleophile is a valid and valuable method to determine pK values of nucleophilic residues in native proteins. The results obtained for elastase (Kaplan et al 1971), chymotrypsin (Kaplan 1972) and calf thymus histones (Malchy and Kaplan 1974) agree well with results obtained from other evidence.…”