Conformational integrity of human alpha-thrombin.
It is known that storage at pH 6 stabilizes thrombin against inactivation. In order to determine whether structural changes accompany this stabilization, the conformation of human alpha-thrombin at pH 6.0 and 7.5 was investigated by chemical modification, difference spectroscopy, circular dichrosim, and thermal stability. It was shown that the CD spectra at the 230-200 nm peptide transition were indistinguishable at the two pH values, indicating no differences in the secondary structure as also indicated by the thermal stability of the enzyme at pH 6.0, 7.4 and 8.3. However, differences were observed in the 300-250 nm aromatic transition suggesting some changes in the microenvironment of the aromatic chromophores. Solvent perturbation in 20% ethylene glycol and 20% dimethylsulfoxide showed that at pH 7.5, 4.3 +/- 0.3 tryptophan and 8.6 +/- 0.4 tyrosine residues were exposed and accessible to the solvent whereas at pH 6.0 these values were 3.6 +/- 0.1 tryptophan and 7.8 +/- 0.4 tyrosine residues. At pH 7.5, 6.0 +/- 0.5 tryptophan residues were found reactive toward dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide while 2.5 +/- 0.3 were found reactive at pH 6.0. Accompanying these structural changes were ultraviolet absorption and CD spectral changes with transition midpoints at pH 6.45 characteristic of histidine ionization. These spectral changes were lost when alpha-thrombin was modified by diethylpyrocarbonate but not by N-alpha-tosyl-L-Lysinechloro-methyl ketone. It is concluded that a second histidine residue, not the active site His-43, is associated with the pH dependent conformational changes at pH 6.0. The ionization of this histidine residue and the accompanying conformational changes could explain the reduced catalytic efficiency and stability of alpha-thrombin at pH 6.
D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate; D006639:Histidine; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013452:Sulfonium Compounds; D013917:Thrombin / Q000032:analysis*; D014107:Tosyllysine Chloromethyl Ketone
D013452:Sulfonium Compounds; D014107:Tosyllysine Chloromethyl Ketone; C024586:dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium; D006639:Histidine; D013917:Thrombin; D004047:Diethyl Pyrocarbonate