Studies on the tryptophan and drug-binding properties of human serum albumin fragments by affinity chromatography and circular dichroism measurements.

After cyanogen bromide digestion of human serum albumin, three fragments, A, B, and C, are obtained. Two different types of a.fEnity columns of Sepharose with L-tryptophan were prepared, one with a free carboxyl group and one with a free amino group. Only Fragment C showed affinity to the Sepharose column with the carboxyl group of tryptophan free, while the column having the amino group free did not extract any fragment from the cyanogen bromide digest. Fragments A, B, and C were separated and studied by circular dichroism. Large parts of the native structure are preserved in the fragments. Human serum albumin contains 45 to 50 % (Y helix, and totally about 35 % is found in the fragments. Fragment A contains about 42 % cy helix and about 15 to 22 % p structure, B contains 22 to 26% a! helix and 9 to 14% /I? structure, and C contains 28 to 36% cy helix and 11 to 32 % /3 structure, the significance of which is discussed. The binding of L-tryptophan and drugs to the different fragments was studied by circular dichroism and equilibrium dialysis; only Fragments A and C were found to be active. The apparent association constants, K,, for the binding of L-[W]tryptophan at pH 9.0 and 10” were 0.16 X 10’ M-’ and 0.17 x lo4 M-’ for Fragments A and C, respectively. The lone tryptophan residue of human serum albumin is found in Fragment C, the importance of which is discussed.