Inactivation of antiplasmin at low pH: evidence for the formation of latent molecules.
Several serine proteinase inhibitors (serpins) are metastable proteins which under certain conditions may undergo conformational changes resulting in the insertion of the reactive centre loop into the so-called Abeta-sheet and hence forming latent molecules. Here we have studied the inactivation of antiplasmin as a function of pH and temperature with time. At decreased pH (4.9-5.8) and at room temperature, antiplasmin activity decreased following first-order kinetics. Analysis by polyacrylamide gel electrophoresis under non-denaturing conditions demonstrated that only minor amounts of polymerized material formed after extensive incubation (4 days) at room temperature. However, on incubation at elevated temperatures (45 or 55 degrees C), a rapid formation of polymerized material was observed. We also demonstrated that antiplasmin inactivated by treatment at pH approximately 5 at room temperature spontaneously slowly regained some activity if incubated in a buffer of neutral pH. Furthermore, by treatment with 4 M guanidinium chloride for about 30 min, followed by dialysis against a neutral phosphate buffer, considerable activity (almost 40%) was regained. Thus, we conclude that antiplasmin, at least partially, at lower temperatures is transformed into a latent form, which could be reactivated, in a similar manner as PAI-1. At increased temperature, however, polymerization seems to be the predominant reason for inactivation.
Wang
Haiyao
H
Department of Clinical Chemistry and Blood Coagulation, Karolinska hospital, Karolinska Institute, Stockholm SE-17176, Sweden.
Pap
Sarolta
S
Wiman
Björn
B
D019281:Dimerization; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D020673:Protein Renaturation; D015843:Serpins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D000979:alpha-2-Antiplasmin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D015843:Serpins; D000979:alpha-2-Antiplasmin; D019791:Guanidine