DETERMINATION OF THE VOLUME CHANGES FOR PRESSURE-INDUCED TRANSITIONS OF APOMYOGLOBIN BETWEEN THE NATIVE, MOLTEN GLOBULE, AND UNFOLDED STATES

The volume change for the transition from the native state of horse he art apomyoglobin to a pressure-induced intermediate with fluorescence properties similar to those of the well-established molten globule or I form was measured to be -70 ml/mol. Complete unfolding of the protei n by pressure at pH 4.2 revealed an upper limit for the unfolding of t he intermediate of -61 ml/mol. At 0.3 M guanidine hydrochloride, the e ntire transition from native to molten globule to unfolded state was o bserved in the available pressure range below. 2.5 kbar. The volume ch ange for the N-->I transition is relatively large and does not correla te well with the changes in relative hydration for these transitions d erived from measurements of the changes in heat capacity, consistent w ith the previously observed lack of correlation between the m-value fo r denaturant-induced transitions and the measured volume change of unf olding for cooperativity mutants of staphylococcal nuclease (Frye et a t. 1996. Biochemistry. 35:10234-10239), Our results support the hypoth esis that the volume change associated with the hydration of protein s urface upon unfolding may involve both positive and negative underlyin g contributions that effectively cancel, and that the measured volume changes for protein structural transitions arise from another source, perhaps the elimination of void volume due to packing defects in the s tructured chains.