Elastase enzyme assays

Enzyme assays of the solutions containing unbound human neutrophil elastase were conducted in pH 7.6 buffer composed of O.IM sodium phosphate, 0.5 M NaCl, and 3.3 % DMSO and subjected to spectrophotomeric measurement of the release of p-nitroaniline at 410 nm from the enzymatic hydrolysis of MeOSuc-Ala-Ala-Pro-Val-pNA (Sigma) (9). The spectrophotometric kinetic assays were performed in a Bio-Rad Microplate Reader (Hercules, CA) with a 96-well format. Two hundred microliter iquots of a elastase solution (0.2 units) were assayed per welt, and 20 microliters of a 475 micromolar substrate solution was added to initiate the enzyme reaction. 

In most of these methods some 10-20 mg of elastin is taken for each tube of the assay the elastase unit is rather large and the assay procedures are expensive in enzyme when they are used to control purification procedures. A much more sensitive method, which measures quantities of elastase of the order of one-hundredth the Banga (1952) unit, was devised by Sbarra et al. (1960). In this assay elastin-agar plates arc used and the method is based on measuring the diameter of the clear zone which is produced by elastase deposited in circular holes cut in the gel. This method of assay is also of particular value in screening microorganisms for the production of elastase. 

AAT can be quantified by all immunochemical methods, with immunoturbidimetry and immunonephelometry the most commonly used methods. Because it constitutes about 90% of the serum inhibition of trypsin or elastase activity against small substrates, such as benzoyl-DL-arginine p-nitroanilide, it can also be semiquantified by the inhibitory capacity of serum for these enzymes however, this assay is not specific for AAT. 

Assay of the isozymers from L rubellus is based on the enzymic reaction with various substrates. F-T and F-III are thought to represent a chymotrypsin-like and a trypsin-like protease, respectively [24], F-II appears to act as a trypsin-like protease or an elastase. Interestingly, F-II1-1 and -2, also with strong caseinolylic activity, have much higher fibrinolytic activity than plasmin as described by Mihara et al. [1,3]... 

From the anti-inflammatory, antipyretic, analgesic, and antioxidant extract of Kageneckia oblonga, Delporte et al. [13] isolated two cucurbitacins which were assayed as potential antioxidants and also as inhibitors of enzymes implicated in inflammatory reactions. Isolated compounds, 23,24-dihydrocucurbitacin F and 3P-( 3-D-glucosyloxy)-16a,23a-epoxy-cucurbitan-5,24-diene-ll-one, Fig. (11), inhibited the production of superoxide anion as well as elastase release in stimulated human neutrophils. In addition, the compounds inhibited both nitrite and... 

A series of ynenol lactones (stmcture 2) were studied as inhibitors of human leukocyte elastase (Tam et al., 1984 Spencer et al., 1986 Copp et al., 1987). Some of the compounds were alternate substrate inhibitors, being hydrolyzed by the enzyme to the reactive I but then deacylat-ing without an inactivation step. However, with the compound 3-benzyl ynenol butyrolactone (stmcture 2, where R = benzyl, R = H), the acyl-enzyme (E-I ) was stable enough to allow the second alkylation step, resulting in inactivated enzyme. All kinetic constants were determined. Continuous assays gave biphasic kinetics, the second minor phase possibly due to the presence of isozymes or enantiomers of the inhibitor. Immediate diffusion-limited inhibition was observed and gave a competitive Ki value of 4.3 0.7 xM. The first phase of inhibition was saturable, and analysis of the rates gave = 0.090 0.007 s , and... 

Treated and untreated gauze samples were submerged in 1 milliliter of buffer containing 1 unit/ mL of human neutrophil elastase. The samples were allowed to incubate for one hour at room temperature after which the gauze samples were removed and placed in an Autovial press filter (Whatman,) to drain unbound buffer and enzyme. The unbound elastase fractions were combined and assayed for elastase activity as described below. 

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