INVOLVEMENT OF SULFHYDRYL-GROUPS IN THE STABLE FLUORESCENT DERIVATIZATION OF PROTEINS BY O-PHTHALALDEHYDE

Treatment of human alpha-1-proteinase inhibitor (alpha-1-PI) with o-ph thalaldehyde (OPA) at pH 8.0 and 25 degrees C, in the absence of added thiol resulted in the formation of a mixed population of fluorescent and non-fluorescent isoindoles. The stoichiometry of isoindole formati on was tentatively calculated to be 6:1 for unreduced alpha-1-PI and 1 0:1 for inhibitor treated with dithioerythritol, implicating not only cysteine but also non-sulfur nucleophilic centres as reaction partners . Despite the apparent involvement of the single cysteine residue in a lpha-1-PI in the over-all derivatization process, the extent of fluore scent derivatization was independent of the redox state of the inhibit or. Hence the fluorescing moiety was not a 1-alkylthio-2-alkyl-substit uted isoindole, as generally observed. The finding that isoindole form ation in proteins is not limited by sulfhydryl content and that fluore scent products may originate from amino acid(s) other than cysteine ca utions against interpreting fluorescent derivatization by OPA as evide nce for cross-linking of lysine to cysteine residues.