ARTICLE pubs.acs.org/Biomac
Effects of Thermal Denaturation on the Solid-State Structure and Molecular Mobility of Glycinin Mickey G. Huson,*,†,‡ Ekaterina V. Strounina,§ Catherine S. Kealley,^,# Manoj K. Rout,†,|| Jeffrey S. Church,‡ Ingrid A. M. Appelqvist,†,|| Michael J. Gidley,§ and Elliot P. Gilbert^ †
CSIRO Food Futures Flagship, North Ryde, NSW 2113, Australia CSIRO Materials Science and Engineering, Belmont, VIC 3216, Australia § The University of Queensland, St. Lucia, QLD 4072, Australia ^ Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia CSIRO Food and Nutritional Science, Riverside Corporate Park, North Ryde, NSW 2113, Australia
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ABSTRACT: The effects of moisture and thermal denaturation on the solid-state structure and molecular mobility of soy glycinin powder were investigated using multiple techniques that probe over a range of length and time scales. In native glycinin, increased moisture resulted in a decrease in both the glass transition temperature and the denaturation temperature. The sensitivity of the glass transition temperature to moisture is shown to follow the GordonTaylor equation, while the sensitivity of the denaturation temperature to moisture is modeled using Flory's melting point depression theory. While denaturation resulted in a loss of long-range order, the principal conformational structures as detected by infrared are maintained. The temperature range over which the glass to rubber transition occurred was extended on the high temperature side, leading to an increase in the midpoint glass transition temperature and suggesting that the amorphous regions of the newly disordered protein are less mobile. 13C NMR results supported this hypothesis.
’ INTRODUCTION The thermal denaturation of proteins involves the disruption of native quaternary, tertiary, and secondary structures, and subsequent reorganization into thermally stable local conformations. Detailed characterization of thermal denaturation in the presence of excess water has been reported,13 but there is little information available on the effects of denaturation on the properties of proteins under limited water conditions. The advantage of studying semicrystalline protein materials at low moisture (20% moisture), the behavior of native and denatured glycinin appear more similar.
ARTICLE
Taken together, the NMR data suggest that, for both native and denatured glycinin, passing from the low moisture glassy state to the high moisture rubbery state involves a simultaneous decrease of mobility in relatively rigid segments (CP-detected) and an increase in mobility of relatively mobile segments (DP-detected). This is consistent with a dual role for water in plasticizing mobile (less structured) segments and in annealing locally structured segments making them more rigid. The differences between native and denatured glycinin are more pronounced at moisture values below Tg with the native form having a broader range of segmental mobility as illustrated by a complex variable contact time response (Figure 8) and some DP-detectable mobile segments (Figure 9). After denaturation, segmental mobility is reduced at low moisture as characterized by faster cross-polarization and lower intensity in DP spectra. These marked differences occur despite the very limited change in average secondary structure proportions as detected by FT-IR after denaturation. A comparison of NMR and FT-IR data suggests that there is no direct relationship between segmental mobility and secondary structure proportions under limited (
