60] Tryptophan involvement in binding sites of proteins and in enzyme-inhibitor complexes as determined by oxidation with N-bromosuccinimide

Abstract

This chapter discusses the tryptophan involvement in binding sites of proteins and in enzyme-inhibitor complexes as determined by oxidation with N-bromosuccinimide (NBS). The effective protection of the tryptophan residues in the avidin-biotin complex and these residues must be present at the biotin-binding site and each biotin molecule blocks or retards the oxidation of four tryptophan residues. Tryptophan in trypsin shows a proportional loss of reactivity toward NBS in the presence of increasing proportions of trypsin-pancreatic inhibitor complex (PTI). Selective modification of tryptophan residues in trypsin and trypsin-PTI complex by NBS is tabulated. In contrast to the natural trypsin inhibitor, a synthetic polypeptide inhibitor enhances tryptophan reactivity in trypsin. The modes of inhibition of the natural and the synthetic inhibitor are essentially different. One third of the tryptophan in the trypsin inhibitor from soybean (STI) can be selectively oxidized at pH 5 by NBS. Only 20–30% of the inhibitory activity is lost. Both the oxidized inhibitor and native STI are rendered completely inactive on urea denaturation.