Electron spin resonance studies of bovine plasma amine oxidase. Probing of the environment about the substrate-liberated sulfhydryl groups in the active site

• Published in: The Journal of biological chemistry 1980-08, Vol.255 (16), p.7621-7626
• Main Authors: Zeidan, H, Watanabe, K, Piette, L H, Yasunobu, K T
• Format: Article
• Language: English
• Subjects: Amine Oxidase (Copper-Containing) - blood; Animals; Binding Sites; Cattle; Chemical Phenomena; Chemistry; Circular Dichroism; Copper; Cysteine; Electron Spin Resonance Spectroscopy; Ethylmaleimide - analogs & derivatives; Hydrogen-Ion Concentration; Protein Conformation; Spin Labels
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43874-X

A series of nitroxide spin-labeled reagents have been employed to explore the environment of the cysteine residues in bovine plasma amine oxidase. When the enzyme was reduced by substrate or phenylhydrazine, 1 essential sulfhydryl residue/subunit was liberated. This cysteine residue was reacted then with the spin label 3-(maleimido-methyl)-2,2,5,5-tetramethyl-1-pyrrolinyloxyl. The ESR spectra of the labeled enzyme derivatives suggested that this essential sulfhydryl residue is located in a pocket, whereas the nonessential sulfhydryl residues are probably located near the surface. By varying the length of the nitroxide spin-labeled N-ethylmaleimide derivatives, it was determined that the liberated essential cysteine residues are in a restricted environment. The ESR spectral data also suggested that the nitroxide radical and the essential copper in the enzyme do not interact with one another. The effect of ionic strength, pH, and urea denaturation on the environment of the essential sulfhydryl residue were also investigated.