Intrinsic fluorescence studies of the kinetic mechanism of unfolding of α-lactalbumin on weakly hydrophobic chromatographic surfaces

• Published in: Journal of Chromatography A 1990-02, Vol.500, p.481-502
• Main Authors: Oroszlan, Peter, Blanco, Rigoberto, Lu, Xiao-Ming, Yarmush, David, Karger, Barry L.
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(00)96087-4

The kinetic mechanism of unfolding of calcium depleted bovine α-lactalbumin adsorbed on two weakly hydrophobic chromatographic surfaces, methyl- and ethyl-polyether phases bonded to porous silica, with a solution phase of 3 M ammonium sulfate at pH 6.3, has been determined using intrinsic fluorescence and liquid chromatography (LC). The adsorbent has been packed into quartz flow cells which are used for both fluorescence measurements and as a microcolumn for LC. The LC measurements revealed two peaks for α-lactalbumin on both phases, the First being folded and the second unfolded. The rate of unfolding was measured to be 1.75 · 10 −3 min −1 on the C1-ether and 7.42 · 10 −3 min −1 on the C2-ether phase. Fluorescence studies revealed a slow change in emission maximum from ca. 330 nm to 350 nm and a 4-fold increase in intensity for the protein adsorbed on the two supports. Variation of fluorescence intensity at a given wavelength revealed biphasic kinetics in which the rate law on the surface was deduced as F ⇌ X → U, where F is the folded form, U an unfolded form and X an intermediate. The normalized emission spectra of the three species were calculated and it was found that there was approximately a 20-nm-red shift in the position of the maximum from F to U. The emission maximum for X was close to U on both columns; however, the normalized intensity for X was between F and U. Activation enthalpies and entropies were determined from the temperature dependence of the microscopic rate constants. The formation of the intermediate on the C1-ether phase was entropy driven whereas on the C2-ether phase it was enthalpy driven. Finally, the solution refolding rates of U desorbed from the two supports were found to be identical. The differences observed in the surface kinetics of unfolding on the two supports are related to the hydrophobic differences of the adsorbents.