Binding Assays for Amino Acids

Abstract Cystine binding protein isolated from Escherichia coli W3092 in high yield was used to develop a binding assay for the disulfide amino acid cystine. Filtration of a mixture of 14Ccystine and cystine binding protein through nitrocellulose filters separated the 14Ccystine-cystine binding protein complex from the unbound cystine. The binding of 14Ccystine-cystine binding protein complexes to the filter was optimal (100%) at pH 4 to 5, and was insensitive to Mg2+ (10 mm), EDTA (10 mm), or concentrations of NaCl to 0.5 m. Bovine serum albumin in excess of 30 µg decreased binding of the 14C complex to the filter. The binding of 14Ccystine to the cystine binding protein was inhibited by lanthionine, much less by diaminopimelic acid, cystathionine, and d-cystine, and was not inhibited by reduced or oxidized glutathione, penicillamine disulfide, cysteic acid, methionine, taurine, homocystine, carboxymethyl cysteine, or the N-ethylmaleimide adduct of cysteine. Nonradioactive cystine competed with 14Ccystine resulting in the expected diminution of the bound radioactivity, permitting the direct calculation of nonradioactive cystine concentrations of unknown samples. With the cystine binding protein assay, the cystine concentrations of human plasma were in the range of 87 to 100% of the values obtained by ion exchange chromatography, and the cystine contents of cultured skin fibroblasts from patients with nephropathic cystinosis were in the range of 89 to 121% of the values obtained by ion exchange chromatography. As little as 10 pmoles of cystine were measured.