Conformational stability of human erythrocyte transglutaminase. Patterns of thermal unfolding at acid and alkaline pH.
Tissue-type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0-9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition-state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent 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 transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-terminal 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-terminal and C-terminal regions interact with each other in determining the structure of the native protein.
Bergamini
C M
CM
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy. bgc@dns.unife.it
Dean
M
M
Matteucci
G
G
Hanau
S
S
Tanfani
F
F
Ferrari
C
C
Boggian
M
M
Scatturin
A
A
D002151:Calorimetry; D002942:Circular Dichroism; D004912:Erythrocytes; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D011503:Transglutaminases / Q000737:chemistry*
D011506:Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins