Gad Feinstein

N-acetyl-(L-Ala)3-p-nitroanilide as a new chromogenic substrate for elastase

Abstract The introduction of a useful new chromogenic substrate for the determination of elastase (EC 3.4.4.7) activity is described. N-acetyl-L-Ala-L-Ala-L-Ala- p -nitroanilide (AcAla3NA) is a new specific elastase substrate whose hydrolysis can be followed spectrophotometrically at 410 nm in a wide pH range. Its rate of hydrolysis by α-chymotrypsin (EC 3.4.4.5) and trypsin (EC 3.4.4.4.) is 0.02% and 0.001% respectively compared to its rate of hydrolysis by elastase. As little as 0.1 μg elastase/ml can be satisfactorily determined. At pH 8, Km = 0.88 mM and kcat = 11.9 sec−1.

Purification of trypsin by affinity chromatography on ovomucoid-sepharose resin

One of the major problems in enzymology is the difficulty in purifying enzymes. The purification is usually done through multiple steps which takes a long time and results in low yields. The usefulness of affinity chromatography in overcoming these difficulties was demonstrated by Fritz et al. [l] . They cross-linked trypsin, cw-chymotrypsin and kallikrein to copolymer of ethylenemaleic acid (EMA), an insoluble carrier introduced by Levin et al. [2] , and used the insoluble enzymes to isolate the corresponding natural inhibitors of these proteases. In later study, Fritz et al. [3 ] fractionated kallikrein and plasmin by chromatography on resin of kallikrein inhibitor crosslinked to EMA. Cuatreccasas et al. [4] cross-linked synthetic inhibitors of several enzymes to the uncharged insoluble carrier Sepharose according to the method of AxCn et al. [5] and used the synthetic inhibitor-Sepharose resins to purify several enzymes by single step of affinity chromatography. Chicken ovomucoid is an egg white protein which is capable of inhibiting bovine trypsin [6] . In this study the method of affinity chromatography was adapted to purification of trypsin by use of chicken ovomucoidSepharose resin. Eighty-five ml of Sepharose 2B (Pharmacia) were activated with CNBr according to Cuatrecases et al. [4] and were coupled to 200 mg chicken ovomucoid (Worthington Biochemical Corp.). The coupling was done by stirring for 24 hr at room temperature the activated Sepharose and the ovomucoid in 250 ml of 0.10 M NaHC03, pH 8.5. After coupling, the ovomucoidSepharose was washed on sintered glass funnel with the following: six times each with 250 ml of 0.10 M NaHCO,, pH 8.5; H20; 0.20 M KCI-HCI,

Proteinase inhibitors in human synovial fluid

Two distinct proteinase inhibitors were detected in human synovial fluid. The two inhibitors were immunologically identical to scrum α1-antitrypsin and α2-macroglobulin. In about 25% of patients with various joint diseases, the α2-macroglobulin inhibitor was found inactive in the synovial fluid though active in their sera. The inactive form of α2-macroglobulin reacted immunologically with monospecific antibody to serum α2-macroglobulin. About 60% of osteoarthritic patients lacked the α2-macroglobulin inhibitory activity. The possibility that enzymatic degradation of articular cartilage is enhanced due to the inactivation of certain proteinase inhibitors, and the possible role of proteinase inhibitors in the pathogenesis of joint diseases are discussed in the light of the reported findings.

Affinity chromatography of biological macromolecules

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Isolation of chicken ovoinhibitor by affinity chromatography on chymotrypsin-sepharose

Abstract Chicken ovoinhibitor was purified from ovomucoid by a new procedure using a single step of affinity chromatography through water-insoluble bovine chymotrypsin-Sepharose. It is capable of inhibiting bovine trypsin and chymotrypsin and has a molecular weight of 52 400.

The digestion of the oxidized B chain of insulin by human neutrophile proteases: Elastase and chymotrypsin-like protease

The specificities of human neutrophile elastase and chymotrypsin-like protease towards oxidized insulin B chain were studied. The neutrophile elastase was found to differ from porcine pancreatic elastase in its specificity towards insulin B chain. The neutrophile elastase preferred mostly valine near the cleaved bond in contrast to pancreatic elastase which preferred alanine as well as valine near the cleaved bond. Human neutrophile chymotrypsin-like protease was found to cleave mostly bonds involving leucine and phenylalanine.

Physiological roles of protein inhibitors

Protein inhibitors of proteases are widespread in nature. They are found in many sources, such as seeds, blood, eggs, and in many types of cells and tissues. Many physiological roles have been attributed to the protein inhibitors. Germination, wound healing, blood clotting, angiogenesis, etc., are some of the normal physiological activities in which protein inhibitors of proteases are involved. Lung destruction during emphysema and cartilage breakdown due to inflammation are two of the pathological conditions in which deficiency of protein inhibitors contributes to tissue breakdown by proteases.

Isolation of alkaline proteinases from Aspergillus oryzae by one-step affinity chromatography on ovoinhibitor-sepharose column

1. 1. Alkaline proteinases from a crude preparation of Aspergillus oryzae were isolated by one-step affinity chromatography on a chicken ovoinhibitor-Sepharose column. The yield of the activity was 55% and the purified fraction had about 300-fold higher specific activity than the starting material. 2. 2. The purified fraction was found to be homogeneous in size but consisted of at least four electrophoretically distinct bands. The amino acid composition was estimated and found to be very similar to other known alkaline proteinases from Aspergillus species, thus indicating a strong genetic relationship. 3. 3. The esterolytic activity of the purified fraction was tested on specific esteratic substrates of pancreatic serine proteases. The purified fraction was active on all 3 substrates, thus indicating a rather broad specificity and the kinetic parameters were almost identical to those of pure alkaline proteinase from Aspergillus sojae. Like the latter, the purified fraction had an extremely high kcat on acetyl tri-l-alanine methyl ester.

Protease Inhibitors in Human Synovial Fluids of Patients with Joint Diseases

The protease inhibitory activities of synovial fluids from patients with various joint diseases were found to be similar to those of healthy patients [1]. The major inhibitory components were found to be electrophoretically and immunologically identical to serum α1, -antitrypsin and α2-macroglobulin. It was found that α2-macroglobulin was present in an inactive form in synovial fluids from most patients suffering from osteoarthritis and some other patients with other joint diseases. Tts presence there was established immunologically but it was incapable of inhibiting trypsin, chymotrypsin or elastase. Apparently the inactive α2 -macroglobulin was either in a form of complex with an unknown component that prevented it from exerting its inhibitory activities or that α2-macroglobulin was inactivated by a local synovial factor, probably a protease.