Yamada H, Matsunaga N, Domoto H, Imoto T
J Biochem. 1986 Jul;100(1):233-41. doi: 10.1093/oxfordjournals.jbchem.a121698.
Dinitrophenylation of hen egg white lysozyme with 2,4-dinitrofluorobenzene (DNFB) was carried out at pH 7-11 and room temperature in order to examine whether dinitrophenylation could be applied to determine the environments of individual amino groups in lysozyme or not. Lightly dinitrophenylated lysozyme was reduced, S-carboxymethylated and then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). All tryptic peptides, which contained dinitrophenylated amino groups (one alpha-amino group, Lys 1(alpha), and six epsilon-amino groups, Lys 1(epsilon), Lys 13, Lys 33, Lys 96, Lys 97, and Lys 116), could be separated and monitored by absorbance measurement at 360 nm on RP-HPLC. The relative reactivities of individual amino groups, determined from the relative peak areas of dinitrophenylated tryptic peptides at 360 nm, were found to be sensitive to the reaction pH and to the presence of the trimer of N-acetyl-D-glucosamine or NaCl. It was concluded that dinitrophenylation of a protein with DNFB followed by peptide analysis by RP-HPLC with detection at 360 nm is a good method for probing the environments of individual amino groups in the protein.
为了研究二硝基苯基化是否可用于确定溶菌酶中各个氨基的环境，在pH 7 - 11和室温条件下，用2,4 - 二硝基氟苯（DNFB）对鸡蛋清溶菌酶进行了二硝基苯基化反应。将轻度二硝基苯基化的溶菌酶进行还原、S - 羧甲基化，然后进行反相高效液相色谱（RP - HPLC）分析。所有含有二硝基苯基化氨基（一个α - 氨基，Lys 1(α)，以及六个ε - 氨基，Lys 1(ε)、Lys 13、Lys 33、Lys 96、Lys 97和Lys 116）的胰蛋白酶肽段，都可以通过RP - HPLC在360 nm处进行吸光度测量来分离和监测。根据360 nm处二硝基苯基化胰蛋白酶肽段的相对峰面积确定的各个氨基的相对反应活性，被发现对反应pH以及N - 乙酰 - D - 葡萄糖胺三聚体或NaCl的存在敏感。得出的结论是，用DNFB对蛋白质进行二硝基苯基化，然后通过RP - HPLC在360 nm处检测进行肽段分析，是探测蛋白质中各个氨基环境的一种好方法。
