Effect of metal ions on the unfolding kinetics of alpha-lactalbumin on weakly hydrophobic surfaces.
The effect of metal ion [Ca2+ and Zn(NH3)4(2+)] on the unfolding kinetics of bovine-alpha-lactalbumin (alpha-LACT) on a weakly hydrophobic chromatographic surface (ethyl polyether phase bonded on porous silica, C2 ether) has been studied using surface intrinsic fluorescence spectroscopy and liquid chromatography. Chromatographic results on the C2 ether phase revealed two peaks for alpha-LACT, the first being the folded and the second an unfolded conformation, as determined by fluorescence spectroscopy. The retention time for the second peak was found to depend on the specific metal additive in the mobile phase. Fluorescence studies showed a slow change in emission maximum from ca. 330 nm to 350 nm and a 5-fold increase in emission intensity for the adsorbed protein in the unfolded state. By following the fluorescence emission intensity at a given wavelength during the unfolding process, biphasic kinetics were observed with the kinetic constants depending on the specific metal-ion additive. In addition, solution refolding rates of the desorbed, unfolded species were measured and found to be consistent with literature refolding rate constants.
Lin
S W
SW
Barnett Institute, Northeastern University, Boston, MA 02115.
Oroszlan
P
P
Karger
B L
BL
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D007477:Ions; D008670:Metals; D008894:Milk Proteins; D007768:Lactalbumin