Disulfide bonds in rat cutaneous fatty acid-binding protein

Unlike other fatty acid-binding proteins, cutaneous (epidermal) fatty acid- binding proteins contain a large number of cysteine residues. The status of the five cysteine residues in rat cutaneous fatty acid-binding protein was examined by chemical and mass-spectrometric analyses. Two disulfide bonds were identified, between Cys-67 and Cys-87, and between Cys-120 and Cys-127 , though extent of formation of the first disulfide bond was rather low in another preparation. Cys-43 was free cysteine, Homology modeling study of t he protein indicated the close proximity of the sulfur atoms of these cyste ine pairs, supporting the presence of the disulfide bonds. These disulfide bonds appear not to be directly involved in fatty acid-binding activity, be cause a recombinant rat protein expressed in Escherichia coli in which all five cysteines are fully reduced showed fatty acid-binding activity as exam ined by displacement of a fluorescent fatty acid analog by long-chain fatty acids. However, the fact that the evolutionarily distant shark liver fatty acid-binding protein also has a disulfide bond corresponding to the one be tween Cys-120 and Cys-127, and that fatty acid-binding proteins play multip le roles suggests that some functions of cutaneous fatty acid-binding prote in might be regulated by the cellular redox state through formation and red uction of disulfide bonds. Although we cannot completely exclude the possib ility of oxidation during preparation and analysis, it is remarkable that a protein in cytosol under normally reducing conditions appears to contain d isulfide bonds.