Abstract
Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the alpha-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced alpha-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce alpha-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.
Highlights
Papain is considered to be the archetype of cysteine proteinases
The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate
It has been shown that heparin can modify the activities of some serine proteinases and its natural inhibitors in vitro (6 – 8) and that heparan sulfate proteoglycans, syndecan-1 ectodomain, and syndecan-4 ectodomain are shed into acute inflammatory wound fluids [9]
Summary
These results strongly suggest that syndecan-1 and syndecan-4 maintain the proteolytic balance in acute wound fluid [10] Syndecans, via their heparan sulfate chain, bind many of the factors that orchestrate the inflammatory response to tissue injury, as well as a variety of extracellular matrix components [9, 10]. Previous results in the literature have shown that papain and cathepsin B are able to bind to laminin of basement membrane [24] These results are consistent with the proposed role of cysteine proteinases in degradation of extracellular matrix components. The bind of papain with glycosaminoglycans may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions, which can regulate its biological functions. A combination of circular dichroism analysis, heparin-Sepharose affinity chromatography, and intramolecularly quenched fluorogenic substrate assays was used to characterize the interaction of papain with glycosaminoglycan
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