Kouno Yousuke, Anraku Makoto, Yamasaki Keishi, Okayama Yoshiro, Iohara Daisuke, Ishima Yu, Maruyama Toru, Kragh-Hansen Ulrich, Hirayama Fumitoshi, Otagiri Masaki
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan; Pharma Daiwa Yuge Pharmacy, Kumamoto City, Japan.
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan.
Biochim Biophys Acta. 2014 Sep;1840(9):2806-12. doi: 10.1016/j.bbagen.2014.04.014. Epub 2014 Apr 22.
Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products.
Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration.
N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present.
The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations.
N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.
辛酸钠（Oct）和N-乙酰-L-色氨酸（N-AcTrp）在白蛋白产品的巴氏杀菌和储存过程中被广泛用作稳定剂。然而，光照会使N-AcTrp发生光降解，形成潜在的有毒化合物。因此，我们研究了N-乙酰-L-蛋氨酸（N-AcMet）与N-AcTrp相比，对白蛋白产品长期稳定性（包括光稳定性）的作用。
对添加和未添加添加剂的重组人血清白蛋白（rHSA）进行4周的光照。通过电子自旋共振光谱法检测不同稳定剂清除活性氧（ROS）的能力。使用荧光胺和蛋白质免疫印迹法通过分光光度法评估羰基含量，而通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、远紫外圆二色光谱和差示扫描量热法检测rHSA的结构。通过超滤法测定结合情况。
发现N-AcMet在光照前后都是一种更优异的ROS清除剂。在N-AcMet存在的情况下，形成的羰基数量最少。根据SDS-PAGE，N-AcMet可稳定rHSA的单体形式，而N-AcTrp在光照期间会诱导rHSA降解。在存在Oct（无论有无N-AcMet）的情况下，rHSA的α-螺旋含量下降最小。当存在N-AcMet时，光照不影响rHSA的变性温度或量热焓。
弱结合的N-AcMet是白蛋白的一种更优异的保护剂，因为它比N-AcTrp是更好的ROS保护剂和结构稳定剂，并且可能对其他蛋白质制剂也有用。
N-AcMet是白蛋白在光照期间的有效稳定剂，而N-Ac-Trp会促进白蛋白的光氧化损伤。
