title: Kinetics of unfolding of proteins on hydrophobic surfaces in reversed-phase liquid chromatography.
abstract: As a continuation of previous studies, we present in this paper measurements on the kinetics of denaturation of papain, soybean trypsin inhibitor and lysozyme on n-butyl-bonded silica gel surfaces used in reversed-phase liquid chromatography (RPLC). In all cases, native and denatured peaks widely separated from one another are observed. The rate constants for denaturation or unfolding are determined by the measurement of the peak area of the native protein as a function of the incubation time that the species spends on the bonded-phase surface. The results reveal that a slow denaturation step occurs with a half-life of ca. 15 min. In addition, studies of denaturation as a function of the amount of 1-propanol in the initial mobile phase suggest an additional unfolding step when the protein comes in contact with the bonded-phase surface. The extent of this latter step decreases as the concentration of 1-propanol increases, further suggesting that 1-propanol sorption on the bonded stationary phase may play a role in this behavior. Other studies are conducted with alpha-chymotrypsinogen, in which injection is made after the start of the gradient. The extent of denaturation is observed to be a function of the organic modifier employed. The results of this paper provide insight into the denaturation process in RPLC and suggest approaches to minimize this behavior.
mesh_terms: D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002853:Chromatography, Liquid / Q000379:methods; D002919:Chymotrypsinogen / Q000302:isolation & purification; D007700:Kinetics; D009113:Muramidase / Q000302:isolation & purification; D010206:Papain / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000302:isolation & purification
chemical_list: D002919:Chymotrypsinogen; D014360:Trypsin Inhibitor, Kunitz Soybean; D009113:Muramidase; D010206:Papain