Tritium-hydrogen exchange in studies of protein structure

Abstract

Tritiated water (THO) has been used in the study of exchangeable H atoms in proteins to provide information on molecular structure. On account of the high sensitivity of detection methods for tritium, it need be used only in tracer amounts and this leads to several advantages over deuterium : (i) there is less risk of conformational changes occurring in the labelled protein, (ii) lower protein concentrations may be used (0.2 to 2.0%), and (iii) rates of exchange are slower. The method has been used to study the T-H exchange of ribonuclease dissolved in THO and the H-T exchange of the N,O-tritiated protein dissolved in H₂O between 0 and 70 °C. The extent of exchange was measured in the former case by T-assay on aliquots of dried protein and in the latter case by T-assay on water samples collected by sublimation. Optimal conditions for sampling, drying, and T-assay are described under which further exchange was minimized. In particular, it was found unnecessary to dry at temperatures above 40 °C. The number of exchangeable H atoms in the ribonuclease molecule was determined as a function of temperature. As reported by Schildkraut and Scheraga (1960) there appears to be a critical temperature below which certain H atoms will not exchange within 24 hr. However, the number of such atoms (50-60) is greater, and the critical temperature (42 °C) lower than previously reported. This discrepancy is discussed as well as the significance of slow and rapid H exchange in relation to protein conformation.