ADSORPTION MECHANISM OF HUMAN SERUM-ALBUMIN ON A REVERSED-PHASE SUPPORT BY KINETIC, CHROMATOGRAPHIC, AND FTIR METHODS

The kinetic behavior of human serum albumin (HSA) adsorbed on a revers ed-phase support was studied. With a phosphate buffer eluent (pH 7.4), the sharp elution front characterizes a fast kinetic adsorption proce ss with a high apparent adsorption rate. In presence of 20% acetonitri le added to the eluent the apparent adsorption rate is about 60 times as low as that found for the first adsorption step in pure buffer. The largest column capacity is found with 20% acetonitrile in buffer; for larger organic solvent contents, a decrease of both the apparent adso rption rate and the column capacity are observed with increasing amoun ts of acetonitrile in the buffer. In order to better understand the ch romatographic behavior of HSA on this type of support, we studied the structural infrared characteristics of the protein in solution. Fourie r transform infrared spectra show that acetonitrile induces some struc tural changes of the protein in solution and competes with alkyl chain s for the interaction with HSA explaining the slow adsorption kinetic process observed in presence of the organic solvent in the eluent. The more compact protein structure found with 20% acetonitrile is correla ted with the larger amount of protein adsorbed at this aqueous buffer- organic solvent composition. (C) 1997 Academic Press.