胞红蛋白的巯基修饰与光谱检测

胞红蛋白（Cygb）是珠蛋白家族中一个近期发现的血红素蛋白，含有血红素辅基，能在卟啉环的铁原子上可逆地结合氧分子，在储存和传递氧中起着重要作用，其C38和C83处含有半胱氨酸。对Cygb上半胱氨酸进行修饰可影响其结合氧的功能。用化学试剂4,4’-二吡啶二硫（4-PDS）、N-乙基马来酰亚胺（NEM）、氧化型谷胱甘肽（GSSG）和二硫苏糖醇（DTT）对纯化的胞红蛋白进行修饰，以分别生成胞红蛋白分子内二硫键（Cygb-SS）、胞红蛋白硫醚键（Cygb-SC）、分子间二硫键（Cygb-SSG）及胞红蛋白自由巯基（Cygb-SH）。修饰效果用4,4’-二吡啶基二硫（4-PDS）光谱法进行检测，该方法通过测量修饰的胞红蛋白中自由巯基的含量来计算修饰率。在Cygb-SS和Cygb-SC样品中检测到巯基浓度比样品浓度的十分之一还低，说明胞红蛋白上的自由巯基已被4-PDS和NEM所占据；Cygb-SSG样品中检测到巯基浓度与蛋白浓度相当，说明Cygb分子中有一个巯基参加了反应，由于Cygb空间位阻的存在，另一个巯基没有改变仍以自由状态存在；Cygb-SH样品中巯基浓度是蛋白浓度的两倍说明一个Cygb分子中含有两个自由巯基。通过分别检测这四种Cygb中自由巯基的含量便可知道其修饰产率。结果表明上述化学试剂均成功地修饰了胞红蛋白，修饰率达到90%以上。将4-PDS光谱法用于检测胞红蛋白化学修饰效果，通过测半胱氨酸巯基浓度验证了该方法的可行性。综上说明4-PDS光谱检测法准确可靠，是对经典Ellman试剂法的补充，更适用于吸收峰在410～420 nm的化合物巯基含量测定。

Sulfhydryl Modification and Spectral Measurement of Cytoglobin

Cytoglobin (Cygb), a member of globin family, is a hemeprotein which was discovered 15 years ago. It contains a prosthetic group heme, which can reversibly bind an oxygen molecule between the iron ion of the porphyrin ring and a histidine of the polypeptide chain and play an important role in storing and delivering oxygen. There are two cysteines in C38 and C83 site of Cygb. Modification of the cysteine on Cygb can affect the oxygen-binding function of Cygb. In this article, chemical reagent 4, 4’- dithiodipyridine (4-PDS), N-ethylmaleimide (NEM), Oxidized Glutathione (GSSG) and Dithiothreitol (DTT) were used for modifying Cygb to intramolecular disulfide bond (Cygb-SS), thioether bonds (Cygb-SC), intermolecular disulfide bond (Cygb-SSG)and free sulfhydryls (Cygb-SH) respectively. The modified effect and product yield were detected by 4, 4’-dipyridine disulfide (4-PDS) spectroscopy method which can calculate modified rate by measuring the free sulfhydryl content in modified Cygb. The concentration of sulfhydryl in Cygb-SS and Cygb-SC samples is lower than one-tenth of the concentration of Cygb, indicating that the free sulfhydryl on the Cygb has been occupied by 4-PDS and NEM; The concentration of sulfhydryl in Cygb-SSG sample is comparable to that of the protein, indicating that one sulfhydryl in Cygb molecule participated in the reaction. Due to the steric hindrance of Cygb, the other sulfhydryl remained unchanged in the free sulfhydryl state; Double the concentration of sulfhydryl in the Cygb-SH sample as compared to the concentration of the Cygb indicates that one Cygb molecule contains two free sulfhydryl. We can know the modified yield by detecting the free sulfhydryl content of the four Cygbs respectively. The results showed that the four chemical reagents successfully modified cysteines on Cygb, and that the product yield reached more than 90%. This article measures the modified effect of Cygb by 4-PDS spectroscopy method and verifies the feasibility of this method by cysteine. In summary, the 4-PDS spectrometry method is accurate and reliable, which is complementary to the classical Ellman’s reagent method, and is more suitable for determining the content of sulfhydryl of compounds with absorption peak at 410~420 nm.