Kinetics of the reaction of thrombin and α2-macroglobulin

Abstract

The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.

Investigation of the selectivity of thrombin-binding aptamers for thrombin titration in murine plasma

“…A2M is a large molecule with a molecular weight of 720 kDa, which is composed of four identical subunits forming a cavity that likely traps thrombin with an association constant of kon=2.5x10 3 M -1 s -1 (Feinman et al, 1985). The sensograms were very similar to those obtained for HCII, and they were interpreted by the gradual association of thrombin to A2M.…”

Limulus α2-macroglobulin. First evidence in an invertebrate for a protein containing an internal thiol ester bond

“…Samples pretreated with methylamine or proteinases are resistant to such autolytic fragmentation (Barrett et al, 1979;Harpel et al, 1979;Ichihara et al, 1981;Janatova & Tack, 1981;Esnard et al, 1985). An additional diagnostic characteristic of thiol ester proteins is that rupture of the thiol ester bond after exposure to proteinases or primary amines results in the appearance of the new free thiol group, which can be titrated with appropriate thiol-reactive reagents (Janatova et al, 1980a,b;Tack et al, 1980;Straight & McKee, 1982;Strickland & Bhattacharya, 1984;Larsson & Bj6rk, 1984;Larsson et al, 1985;Feinman et al, 1985). In the present paper we report evidence that a functional homologue of c2-macroglobulin from the plasma of the American horseshoe crab Limulus polyphemus (Quigley & Armstrong, 1983 possesses an internal thiol ester bond.…”

“…Thiol groups of a2-macroglobulin were quantified with 4,4'-dipyridyl disulphide by monitoring the increase in absorbance at 324 nm (Feinman et al, 1985). The molar absorption coefficient of the reaction product of 4,4'dipyridyl disulphide with cysteine was determined to be 1.68 x IO'm4 M-l-cm-'.…”

“…Thiol ester proteins are activated by proteolytic attack at a defined site situated some distance from the thiol ester group (Harpel, 1973;Starkey & Barrett, 1977;Barrett et al, 1979;Swenson & Howard, 1979a;Sottrup-Jensen et al, 1981b). The activated thiol ester group can mediate covalent attachment to acceptor molecules by ester or amide bonding to the y-carbonyl group of the glutamic acid residue (Swenson & Howard, 1979b;Law et al, 1979Law et al, , 1980aSalvesen & Barrett, 1980;Campbell et al, 1980;Pangburn & Muller-Eberhard, 1980;Sottrup-Jensen et al, 1980, 198 la;Salvesen et al, 1981 ; Gadd & Reid, 1981;Howard, 1981;Van Leuven et al, 1981;Wang et al, 1981 a,b;Wu et al, 1981;Feinman et al, 1985). The thiol ester group of the non-proteolysed native protein also is sensitive to nucleophilic attack at the y-carbonyl group of the glutamic acid residue by small primary amines such as methylamine and ammonia (Harpel, 1976;Barrett et al, 1979;Swenson & Howard, 1979b, 1980Sottrup-Jensen et al, 1980;Tack et al, 1980;Howard, 1980Howard, , 1981Salvesen et al, 1981;Harrison et al, 1981;Starkey et al, 1982;Dangott & Cunningham, 1982;Larsson & Bj6rk, 1984;Larsson et al, 1985;Esnard et al, 1985).…”

The Yin–Yang of thrombin and activated protein C

“…Indeed, thrombin generation is kept in close check under physiological conditions with a short half‐life of only 15 s (Jesty, 1986). It is rapidly inactivated by different inhibitors within the family of serine protease inhibitors (SERPINS), such as antithrombin (Rosenberg & Rosenberg, 1984), α 1 ‐antitrypsin (α 1 AT) and heparin co‐factor II (Derechin et al , 1990), as well as by α 2 ‐macroglobulin (α 2 M) (Feinman et al , 1985). At the boundaries of tissue injury, any excess thrombin is then bound by the receptor thrombomodulin (TM) on intact endothelial surfaces.…”