Study of the denaturation/aggregation behaviour of whole porcine plasma and its protein fractions during heating under acidic pH by variable-temperature FTIR spectroscopy

Abstract

Variable-temperature (VT) Fourier transform infrared (FTIR) spectroscopy was employed to examine changes in secondary structure of whole plasma proteins as well as of plasma protein fractions (serum, serum albumin and globulins) upon heating at pH 4.5 and to establish their kinetics of thermally induced protein aggregation through formation of non-native intermolecular beta-sheets. A detailed analysis of the amide I′ band in the VT-FTIR spectra indicated that plasma proteins were more thermally sensitive at pH 4.5 than at pH 7.5 both when found as mixtures and in monomolecular systems, with the thermal aggregation being strongly enhanced under acidic conditions, particularly in the case of serum albumin. Comparison of the spectral changes of plasma and serum (fibrinogen-depleted plasma) during heating indicated that fibrinogen has no role in protein aggregation under acidic conditions, in contrast to findings at pH 7.5. Considering the particular characteristics of the different plasma proteins, the strong predominance of positive charges in the plasma as a whole at pH 4.5 along with the effects of these pH conditions on the conformation of fibrinogen could be suggested as the main factors responsible for the lack of a contribution by fibrinogen to protein aggregation. Moreover, 2D correlation spectroscopy indicated that the sequence of structural changes occurring during heating was practically identical among the different protein fractions examined and completely different from that established at pH 7.5, with the native beta-sheets being now more heat-sensitive than the alpha-helical structures and with protein aggregation through the formation of intermolecular beta-sheets beginning after native beta-sheets started to unfold.