Fluorescence polarization studies on the interaction of active site modified chymotrypsins with alpha1-protease inhibitor.
Fluorescence polarization has been used to study the interaction of human alpha1-protease inhibitor (alpha1 PI; also called alpha1-antitrypsin) with two active site modified chymotrypsins (CT), dehydroalaninyl-195-alpha-CT (AnhCT) and N-methylhistidinyl-57-alpha-CT (MeCT). For the reaction of the fluorescein-labeled AnhCT (FAnhCT) with alpha 1 PI (Pi type MM, the predominant allelic form), a Kassoc of 1.8 x 10(7) M-1 was obtained by Scatchard analysis, which also indicated 1.3 binding sites. An alternate analysis using a direct dissociation plot, which assumes 1:1 binding, gave a Kassoc of 2.2 x 10(7) M-1. Fluorescein-labeled MeCT (FMeCT) binds somewhat more weakly to alpha  PT (Kassoc = 1.2 x 10(6) M-1; 0.87 binding site). Similar results were obtained by using the proflavin displacement method to determine the binding constant for MeCT with alpha 1 PI (Kassoc = 1.0 x 10(6) M-1). With alpha 1 PI (ZZ type) in which the serum level is reduced and there is a strong tendency to develop chronic obstructive pulmonary disease, the Kassoc found by the fluorescence polarization method was similar to that for alpha 1 PI (MM type) for both CT derivatives. Alpha 1 PI (MM type), modified by oxidation with N-chlorosuccinimide, shows a reduced binding affinity for FAnhCT (Kassoc = 6.5 x 10(5) M-1) and no measurable binding with FMeCT (Kassoc less than 1 x 10(4) M-1). Previous studies have demonstrated that bovine CT forms very stable complexes with alpha 1 PI. In contrast, complexes formed with both active site modified CT derivatives undergo rapid dissociation as shown by the drop in the polarization value on dilution or on the addition of excess unlabeled chymotrypsin derivative. This weakened association suggests that, for reaction with alpha 1 PI, the enzyme active site serine is important in stabilizing the enzyme-inhibitor complex.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D000515:alpha 1-Antitrypsin / Q000494:pharmacology*
D000515:alpha 1-Antitrypsin; D002918:Chymotrypsin