STRUCTURAL INVESTIGATION OF TRANSGLUTAMINASE BY FOURIER-TRANSFORM INFRARED-SPECTROSCOPY

The secondary structure of transglutaminase was investigated by Fourier tranform infrared spectroscopy. Spectra of the protein in both H2O and (H2O)-H-2 were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide-I band. The quantitative analysis of the amide-I-band components was made by a curve-fitting procedure. The protein was studied in the absence and in the presence of 1 mM GTP, 1 mM Ca2+ and 1 mM GTP/1 mM Ca2+. The quantitative analysis of infrared spectra revealed that no remarkable changes in the secondary structure of the enzyme are induced by GTP, Ca2+ or Ca2+/GTP. Major changes, however were observed in the thermal-denaturation behavior of the protein. The protein showed maximum of denaturation at temperatures over 50-55-degrees-C in the absence or in the presence of 1 mM Ca2+ and over 55-60-degrees-C in the presence of 1 mM GTP or 1 mM Ca2+/1 mM GTP. The results obtained indicate that GTP induces a stabilization of the tertiary structure of the enzyme, even in the presence of 1 mM Ca2+. The thermal denaturation patterns of the protein suggest the occurrence of Ca2+-dependent aggregation.