alpha-chymotrypsin and 4-nitrobenzenesulfonyl-fluoride

The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin.

Topics: Amino Acid Sequence; Avidin; Bacterial Proteins; Binding Sites; Biotin; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Chymotrypsin; Hydrolysis; Indicators and Reagents; Molecular Sequence Data; Nitrobenzenes; Peptide Fragments; Streptavidin; Trypsin; Tyrosine

Topics: Amino Acids; Animals; Cattle; Chromatography, High Pressure Liquid; Chymotrypsin; Deoxyribonucleases; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Nitrobenzenes; Pancreas; Peptide Fragments; Trypsin; Tyrosine