Location of the Indole Binding Site in Human Serum Albumin

Abstract

Abstract Human serum albumin was labeled with N-bromoacetyl-l-tryptophan (attached at the 3 position of a histidyl residue), pyridoxal 5'-phosphate (attached to the e-amino group of lysyl residues) and 1-dimethylaminonaphthalene-5-sulfonyl chloride (attached to e-lysyl and tyrosyl-OH residues). Binding studies with these preparations were conducted with N-acetyl-l-tryptophan to determine the extent of blockage of the indole binding site by these reagents. At labeling conditions of 1:1 molar ratio of labeling agents to albumin, 25 to 65% blockage of the indole binding site occurred. When labeling was conducted in the presence of indolepropionate, the labeling was much reduced at the indole binding site, confirming the specificity of the labeling agents. Following the procedures used by McMenamy, Dintzis, and Watson for unlabeled human serum albumin ((1971) J. Biol. Chem. 246, 4744) CNBr fragmentation of the labeled albumins was conducted and the three major fragments were isolated. Sixty-five per cent or more of the attached labels in each preparation were found in Fragment C (mol wt, ∼18,000). At least one of the labeled positions in this fragment (but not all) was effective in blocking the indole binding site. A tyrosyl-OH group, which was located in Fragment A (mol wt, ∼33,000) and which reacted to a limited extent with 1-dimethylaminonaphthalene-5-sulfonyl chloride also influenced binding at the indole site. The diversity of the labeling agents, the diversity of the groups to which the labels were attached, and other physical-chemical considerations lead to the conclusion that the site is very flexible and adaptable. The evidence supports the positioning of the indole binding site between Fragments A and C as they exist in whole albumin. The dominance of the labeling positions in Fragment C also leads to the conclusion that this fragment is the major locus for ligand binding in albumin.