Effect of temperature on the secondary structure of β-lactoglobulin at pH?6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis

摘要

pPrevious CD measurements of changes in the conformation of β-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 °C. New CD measurements are reported at temperatures up to 80 °C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 °C with a ZnSe circle cell and a single simplified model of the substructure of the amide I′ band was used to give the fractional contents of β-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of β-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 °C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 °C a partly unfolded state is formed, possibly by destabilization of the intermolecular β-strand I and the loss of the main helix, but it is not a classical molten globule transition./p