Hatakeyama T, Yamasaki N, Funatsu G
J Biochem. 1986 Sep;100(3):781-8. doi: 10.1093/oxfordjournals.jbchem.a121771.
The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.
蓖麻毒素D低亲和力（LA-）结合位点的糖结合能力通过N-溴代琥珀酰亚胺（NBS）氧化被消除，而在乳糖存在的情况下，最终被氧化的色氨酸残基数量减少了1摩尔/摩尔，并且糖结合能力得以保留（Hatakeyama等人，（1986年）《生物化学杂志》99卷，1049 - 1056页）。基于这些发现，确定了位于蓖麻毒素D的LA结合位点的色氨酸残基。分别在有和没有乳糖存在的情况下用NBS修饰的两种蓖麻毒素D衍生物被分离成它们的组成多肽链（A链和B链）。发现被修饰的一个或多个色氨酸残基存在于B链中，而不存在于A链中。从赖氨酰内肽酶和胰凝乳蛋白酶消化产物中，通过在Bio - Gel P - 30上的凝胶过滤和高效液相色谱法分离出含有氧化色氨酸残基的肽段。对含有氧化色氨酸的肽段的分析表明，在蓖麻毒素D的无活性衍生物中，B链上第37、93和160位的三个色氨酸残基被氧化，其中LA结合位点的糖结合能力通过NBS氧化被消除。另一方面，在乳糖存在下被修饰的蓖麻毒素D活性衍生物中，被修饰的残基被确定为第93和160位的色氨酸，这表明与乳糖结合后，B链上第37位的色氨酸残基受到保护不被NBS氧化。从这些结果表明，B链上第37位的色氨酸是蓖麻毒素D的LA结合位点上糖结合的必需残基。
