  An approach has been proposed to characterize the competitive association of
D2O and to study the heterogeneity of hydration water adsorbed by the protein,
immunoglobulin G, using methodology for determining of non-freezing water in
mixed H2O-D2O protein solutions by low-temperature 1H NMR technique. Direct
data on the numbers of deuteriums adsorbed by immunoglobulin G and isothermals
of water (D2O) sorption by the protein for solution hydration conditions were
obtained. The preferential binding of D2O as well as the isotopic effect of low
D2O concentrations was simply confirmed using this method. The shape of the
isothermals, similar to that for polymolecular adsorption, demonstrates
relative changes in the fractions of heavy water isotope bound to different
groups of protein atoms on decreasing temperature in frozen solution. At -35
deg C the major fractions attached to charged and polar atomic groups appear to
be related as 2/3. The adsorption curves indicate the direct relationship of
non-freezing water to interface water in protein powders.
