Abstract
We previously reported a method of fluorometric determination of elastase activity after separating other protein and enzymatic reaction products by trichioroacetic acid.
On the other hand, Substrate of soluble elastin (α-, β-elastin and elastase solubilized elastin) and other protein was not separated by the deproteinizing procedure. We improved above method separating substrate and enzymatic reaction products by dialysis method.
As a result, we could get Km values of porcine pancreatic elastase, human pancreatic elastases 1 and 2 toward α-elastin, which were 0.51, 2, 84, 2.38 respectively and those toward β-elastin were 3.70, 2.24, 2.35 respectively. It may be suggested that porcine pancreatic elastase is tend to digest α-elastin rather β-elastin, but human pancreatic elastases 1 and 2 tend to digest similarly both elastins. Furthermore, we could find that elastase solubilized elastin was also digested by trypsin, chymotrypsin, collagenase and pronase. But insoluble elastin and α-, β-elastin as the substrate were not digested by the above enzymes.
It has been suggested that insoluble elastin is primarily solubilized by elastase and then soluble elastin fragment is digested by other proteolytic enzymes.
The elastase assay presented here should be usefull for the study of elastin metabolism under normal and various disease states.
