Abstract
We recently reported that Serratia 56 K protease is inhibited by plasma α2 macroglobulin (α2M) temporarily and by chicken egg white ovomacroglobulin (ovoM) continuously (Molla, A. et al. (1986) Infect. Immun. 53, 522-529). The inhibition of this protease is almost complete with ovoM whereas it is incomplete with α2M, although these two macroglobulins show homology and many similarities. In the present study we determined the apparent numbers of binding sites and binding constants for the two macroglobulins by means of the fluorescence polarization method using FITC-labeled 56 K protease. The time courses of complex formation of 56 K protease with α2M and ovoM were different; with ovoM it was complete within 5 min while with α2M 150 min was required. Their apparent molecular volumes were also different; the fluorescence polarization value of the E/I complex was 18.7%. larger with ovoM than with α2M. The association constants obtained on Scatchard plot analysis with 56 K protease and α2M or ovoM were 0.33×107M-1 and 1.09×107M-1, respectively. One molecule of each of these macroglobulins binds 1.13 and 1.35 molecules of 56 K protease, respectively. Upon E/I complex formation, an increase in amino groups due to proteolysis was noted in both cases, but more progressive proteolysis was observed in the case of α2M. Furthermore, when the 56 K protease was inactivated through the depletion of Zn atoms, complex formation did not occur.
