Drug-binding and other physicochemical properties of a large tryptic and a large peptic fragment of human serum albumin

Abstract

The diazepam-binding behaviour of a large tryptic and a large peptic fragment of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate the primary diazepam-binding site on the albumin molecule. The analytical set-up of the FPLC® was used to find the optimum experimental conditions for isolating the fragments. Conventional columns with a 100-fold higher loading capacity than the analytical FPLC ® columns were used to isolate large amounts of the fragments. The isolation procedure for the tryptic fragment (45 kDa, domains two and three of the albumin structure) is described in this paper. The description of the isolation procedure for the peptic fragment (46 kDa, domains one and two of the albumin structure) is published elsewhere (Bos, O.J.M., Fischer, M.J.E., Wilting, J. and Janssen, L.H.M. (1988) J. Chromatogr. 424, 13–21). The induced ellipticity of the diazepam-fragment complexes as well as the affinity of diazepam to the fragments turned out to be pH dependent. This pH dependence occurs in the region of the neutral to base transition of the albumin molecule. Difference CD-spectra of the proteins showed that the tryptic fragment and albumin have similar diazepam-binding properties, whereas the peptic fragment has different diazepam-binding properties. This result is in line with our equilibrium dialysis experiments which showed that the affinity of diazepam to the tryptic fragment and to albumin is of the same order of magnitude, whereas the affinity of diazepam to the peptic fragment is several orders of magnitude lower. On the basis of these results, it can be concluded that the tryptic fragment contains the primary diazepam-binding site and the peptic fragment one or more secondary diazepam-binding sites. This means that at least the main part of the primary diazepam-binding site is located in domain three of the albumin structure.