The Interaction Of Alpha(2)macroglobulin With Trypsin, Chymotrypsin, Plasmin, And Papain.

Although considerable efforts have been made to understand and describe the mechanism of interaction between human plasma (alpha)(,2)macroglobulin and a wide variety of proteinases, there is not general agreement within the literature concerning even the most basic aspects of this unique proteinase-inhibitor reaction. This dissertation describes a purification procedure for highly active human plasma (alpha)(,2)macroglobulin and investigations which sought to help clarify some questions about the (alpha)(,2)macroglobulin-proteinase interaction. The proteinase:(alpha)(,2)macroglobulin molar trapping ratios for trypsin (2:1), chymotrypsin (between 1.4:1 and 1.8:1 depending on experimental conditions), and papain (2:1) were determined. The intrinsic fluorescence increase which occurs in the (alpha)(,2)macroglobulin molecule upon interaction with proteinases or small molecule amines was analyzed and the amount of fluorescence increase was found to be directly proportional to added proteinase (up to saturation) for the four proteinases studied. Contrariwise, the release of free sulfhydryl groups from the thioesters of (alpha)(,2)macroglobulin which occurs upon interaction with proteinases was found to be non-stoichiometric (formation of 1:1 complexes between (alpha)(,2)macroglobulin and trypsin or chymotrypsin resulted in release of all four (alpha)(,2)macroglobulin sulfhydryl groups). In order to better understand these incongruous observations, the kinetics of the fluorescence increase and sulfhydryl release reactions of (alpha)(,2)macroglobulin-proteinase reaction mixtures were studied and the following results were obtained: (1) for the four proteinases studied, all reactions appeared to be biphasic, each phase being first order; and (2) the fluorescence increase and sulfhydryl release reactions occurred with the same rate constants (trypsin: 8 seconds('-1) (fast phase) and 1 seconds('-1) (slow phase); chymotrypsin: 4 seconds('-1) and 0.3 seconds('-1); plasmin: 1 seconds('-1) and 0.1 seconds('-1); and papain (fluorescence data only): 10 seconds('-1) and 2 seconds('-1). As a result of these studies, a model mechanism for the formation of the 1:1 trypsin:(alpha)(,2)macroglobulin complex is proposed. The applicability of this model to formation of the 2:1 trypsin:(alpha)(,2)macroglobulin complex as well as other (alpha)(,2)macroglobulin-proteinase complexes is then considered.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128012/2/8712134.pd