Single microassay for matrix degrading enzymes.
Matrix degrading enzymes are implicated in several disease processes such as abdominal aortic aneurysms and emphysema, however, monitoring proteolytic activity in a single assay is not well-established. Numerous assays have been developed to measure matrix degrading enzymes, which use artificial substrates or substrates derived from natural substrate protein. We have recently developed an assay for elastolytic activity based on the detection of primary amines, using trinitrobenzene sulfonic acid (TNBSA), following the digestion of succinylated elastin. The assay is also versatile enough to allow the detection of other proteases through the use of succinylated substrate specific for given protease. In order to improve the sensitivity and versatility of the assay we have refined the buffer conditions (PBS pH 7.2/1 mM CaCl2) to provide a 60 % increase in sensitivity to elastolytic activity and also formulated a substrate mixture containing succinylated elastin, collagen and gelatin. The combination of a substrate mixture and an optimum buffer will allow a spectrum of enzymes to be detected in a single reaction, providing a more complete picture of total proteolytic activity in a biological sample. This assay may also provide a tool to use proteolytic activity as a marker to monitor pathologic conditions involving matrix turn-over.
Long Island Jewish Medical Center, Long Island campus for the Albert Einstein College of Medicine, 270-05 76th Avenue, New Hyde Park, New York 11042, USA. mathrubu@lij.edu
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D002021:Buffers; D002122:Calcium Chloride / Q000737:chemistry; D004796:Clinical Enzyme Tests / Q000379:methods*; D003094:Collagen / Q000378:metabolism; D003240:Connective Tissue Diseases / Q000175:diagnosis*; D004549:Elastin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism; D006801:Humans
D002021:Buffers; D005780:Gelatin; D003094:Collagen; D004549:Elastin; D010450:Endopeptidases; D002122:Calcium Chloride