A Low-temperature 1H NMR Study of H2O and D2O Associated Competitively with Immunoglobulin G in Solution

TL;DR

This study uses low-temperature 1H NMR to analyze how D2O and H2O compete for binding sites on immunoglobulin G, revealing preferential D2O binding and heterogeneity in hydration water at sub-zero temperatures.

Contribution

It introduces a methodology for characterizing competitive D2O/H2O association with proteins using low-temperature 1H NMR, providing direct data on isotope adsorption and hydration heterogeneity.

Findings

D2O shows preferential binding to immunoglobulin G.

Isothermals suggest polymolecular adsorption behavior.

Heavy water binds mainly to charged and polar groups at low temperatures.

Abstract

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.