Shechter Y, Burstein Y, Patchornik A
Biochemistry. 1975 Oct 7;14(20):4497-503. doi: 10.1021/bi00691a025.
Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).
在中性和略碱性pH条件下，肽和蛋白质中的甲硫氨酸残基会被温和的氧化剂如氯胺-T和N-氯代琥珀酰亚胺氧化为甲硫氨酸亚砜。使用氯胺-T时，半胱氨酸也会被氧化为胱氨酸，但其他氨基酸不会被修饰；使用N-氯代琥珀酰亚胺时，色氨酸也会被氧化。在肽和变性蛋白质中，所有甲硫氨酸残基都会被定量氧化，而在天然蛋白质中，只有暴露的甲硫氨酸残基会被修饰。甲硫氨酸残基的氧化程度通过用溴化氰对未氧化的甲硫氨酸残基进行定量修饰来确定（而甲硫氨酸亚砜残基保持完整），然后进行酸水解和氨基酸分析。甲硫氨酸被测定为高丝氨酸，甲硫氨酸亚砜被还原回甲硫氨酸。通过用溴化氰切割未氧化的甲硫氨酰肽键，然后对新的氨基末端氨基酸进行定量端基分析（通过自动测序仪），以类似的方式确定氧化位点。
