Involvement of sulfhydryl groups in the stable fluorescent derivatization of proteins by o-phthalaldehyde

• Published in: Analyst (London) Vol. 120; no. 4; pp. 1087 - 1090
• Main Authors: KURALAY, A, ORTAPAMUK, O, YILMAZ, S, SÜMER, N, ÖZER, I
• Format: Journal Article
• Language: English
• Published: Cambridge Royal Society of Chemistry 01.04.1995
• Subjects: alpha 1-Antitrypsin - analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; o-Phthalaldehyde; Oxidation-Reduction; Proteins - analysis; Sulfhydryl Compounds - analysis; Vertebrata; Mammalia; Stability; Bovine; Fluorescence; Enzyme inhibitor; α1-Antiproteinase; Artiodactyla; Ungulata; Biological activity
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/AN9952001087

Treatment of human alpha-1-proteinase inhibitor (alpha-1-PI) with o-phthalaldehyde (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 formation was tentatively calculated to be 6:1 for unreduced alpha-1-PI and 10:1 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 alpha-1-PI in the over-all derivatization process, the extent of fluorescent derivatization was independent of the redox state of the inhibitor. Hence the fluorescing moiety was not a 1-alkylthio-2-alkyl-substituted isoindole, as generally observed. The finding that isoindole formation in proteins is not limited by sulfhydryl content and that fluorescent products may originate from amino acid(s) other than cysteine cautions against interpreting fluorescent derivatization by OPA as evidence for cross-linking of lysine to cysteine residues.