Influence of saccharides on the stabilization of frozen hemeprotein

The purpose of this study is to evaluate the stabilization effects of sucrose, glycerol and trehalose on Hb as a representative of hemeprotein during freezing. The indicators were the amount of precipitate of the denatured protein, metHb formation by oxidation and Hill constant derived from the oxygen binding ability. The results obtained were as follows: The amount of the denatured precipitate was 3.6%, and metHb was 51% in control Hb at seventh month of freezing. The oxyHb-pO2curve tended to be from the sigmoid to the hyperbola with increasing metHb. The Hill constant decreased from 3.0 to 1.4. No precipitate was observed in the samples containing more than 0.4% sucrose, glycerol or trehalose during 7 month's freezing. On addition of sucrose and glycerol of more than 1.6% or trehalose of 1.6-3.0%, the allosteric function of Hb could be kept even being frozen for a long-term. From this study, it is clear that sucrose, glycerol and trehalose have stabilization effects on hemeproteins. The mechanism was suggested in which the carbohydrates selectively form a pseudo-hydrated layer with surface water surrounding protein and stabilize the tertiary structure of hemeprotein molecule, resulting in protection against heme oxidation.