Structural requirements for drug binding to site II on human serum albumin.

Structure-activity relationships for binding at site II on human serum albumin have been investigated using drugs, fatty acids, and aliphatic amines with chain lengths C-3 to C-18. A negative charge is not required for binding provided a strongly electronegative center is present. For example, diazepam, a basic drug that exists mainly in the un-ionized form at neutral pH, also binds with high affinity to site II. However, aliphatic amines (pKa values 10-11) with chain lengths C-3 to C-12 did not displace markers from either site I or II, showing that the presence of a positive charge precludes binding at these sites. Short-chain fatty acids, C-3 to C-5, did not displace marker drugs or fluorescent probes from either site I or II when added at equimolar ratios with albumin. Displacement of site II (but not site I) markers occurred with medium-chain fatty acids, C-7 to C-11, and was maximal at C-10. Fatty acids with chain lengths C-10 to C-18 caused an enhancement of fluorescence of dansylamide bound to site I, the maximal effect occurring with C-12. Both arylpropionic acid nonsteroidal anti-inflammatory drugs and medium-chain fatty acids binding at site II had molecular lengths within the range 11-16 A. The effect of hydrophobicity (and/or molecular length) on binding affinity was much more marked with the arylpropionic acids than with the fatty acids, suggesting that bulkier aromatic molecules form more effective interactions at the binding site. The results suggest that site II is a hydrophobic cleft about 16 A deep and about 8 A wide in the albumin molecule with a cationic group located near the surface.