{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T11:23:55Z","timestamp":1758281035476},"reference-count":0,"publisher":"Portland Press Ltd.","issue":"2","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[1974,8,1]]},"abstract":"<jats:p>A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan -69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pKa of the acidic limb of the pH profile by more than 1 pH unit for both kcat. and kcat.\/Km. The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan -69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan -177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pKa of 4.2. Histidine -159, which is in close contact with tryptophan -177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan -177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pKa determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N\u2013benzyloxycarbonylglycylglycine amide and N\u2013benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan -177 were shown to be structural features of the S2 and S1\u2032 subsites respectively.<\/jats:p>","DOI":"10.1042\/bj1410503","type":"journal-article","created":{"date-parts":[[2015,8,10]],"date-time":"2015-08-10T19:54:15Z","timestamp":1439236455000},"page":"503-515","update-policy":"http:\/\/dx.doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":38,"title":["A kinetic and fluorimetric investigation of papain modified at tryptophan -69 and -177 by <i>N<\/i>\u2013bromosuccinimide"],"prefix":"10.1042","volume":"141","author":[{"given":"Gordon","family":"Lowe","sequence":"first","affiliation":[{"name":"The Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QY, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alan S.","family":"Whitworth","sequence":"additional","affiliation":[{"name":"The Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QY, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/141\/2\/503\/562526\/bj1410503.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/141\/2\/503\/562526\/bj1410503.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,26]],"date-time":"2021-11-26T16:21:52Z","timestamp":1637943712000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/141\/2\/503\/9519\/A-kinetic-and-fluorimetric-investigation-of-papain"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1974,8,1]]},"references-count":0,"journal-issue":{"issue":"2","published-print":{"date-parts":[[1974,8,1]]}},"URL":"https:\/\/doi.org\/10.1042\/bj1410503","relation":{},"ISSN":["0264-6021"],"issn-type":[{"value":"0264-6021","type":"print"}],"subject":[],"published":{"date-parts":[[1974,8,1]]}}}