Conformational stability of human erythrocyte transglutaminase - Patterns of thermal unfolding at acid and alkaline pH

Tissue-type transglutaminase is irreversibly inactivated during heat-treatm ent. The rate of inactivation is low at pH 7.5; it increases slightly at ac id pH (6.1) but much more at alkaline pH (9.0-9.5), suggesting that specifi c effects take place in the alkaline range, possibly in relation,to decreas ed stability of the transition-state intermediate as pH is raised above 9.0 . Differential scanning-calorimetry experiments indicate that thermal unfol ding of the protein occurs with two separate transitions involving independ ent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, :by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH we noted that transglutamin ase was unfolded via different pathways at the different pH values consider ed. At acid;:pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so; at alkaline pH, agg regation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of cruci al lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-t erminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C-terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N-termina l and C-terminal regions interact with each other in determining the struct ure of the native protein.