Otsuka Makoto, Fukui Yuya, Ozaki Yukihiro
Research Institute of Pharmaceutical Sciences, Musashino University, Shinmachi, Nishi-Tokyo, Japan.
Colloids Surf B Biointerfaces. 2009 Mar 1;69(2):194-200. doi: 10.1016/j.colsurfb.2008.11.016. Epub 2008 Nov 27.
The purpose of this study was to evaluate the enzymatic stability of colloidal trypsin powder during heating in a solid-state by using Fourier transform infrared (FT-IR) spectra with chemoinformatics and generalized two-dimensional (2D) correlation spectroscopy. Colloidal crystalline trypsin powders were heated using differential scanning calorimetry. The enzymatic activity of trypsin was assayed by the kinetic degradation method. Spectra of 10 calibration sample sets were recorded three times with a FT-IR spectrometer. The maximum intensity at 1634cm(-1) of FT-IR spectra and enzymatic activity of trypsin decreased as the temperature increased. The FT-IR spectra of trypsin samples were analyzed by a principal component regression analysis (PCR). A plot of the calibration data obtained was made between the actual and predicted trypsin activity based on a two-component model with gamma(2)=0.962. On the other hand, a 2D method was applied to FT-IR spectra of heat-treated trypsin. The result was consistent with that of the chemoinformetrical method. The results for deactivation of colloidal trypsin powder by heat-treatment indicated that nano-structure of crystalline trypsin changed by heating reflecting that the beta-sheet was mainly transformed, since the peak at 1634cm(-1) decreased with dehydration. The FT-IR chemoinformetrical method allows for a solid-state quantitative analysis of the bioactivity of the bulk powder of trypsin during drying.
本研究的目的是通过结合化学信息学和广义二维（2D）相关光谱的傅里叶变换红外（FT-IR）光谱，评估胶态胰蛋白酶粉末在固态加热过程中的酶稳定性。使用差示扫描量热法对胶态结晶胰蛋白酶粉末进行加热。通过动力学降解法测定胰蛋白酶的酶活性。用FT-IR光谱仪对10个校准样品集的光谱进行三次记录。随着温度升高，FT-IR光谱在1634cm(-1)处的最大强度以及胰蛋白酶的酶活性均降低。通过主成分回归分析（PCR）对胰蛋白酶样品的FT-IR光谱进行分析。基于γ(2)=0.962的双组分模型，绘制了实际和预测的胰蛋白酶活性之间的校准数据图。另一方面，将二维方法应用于热处理胰蛋白酶的FT-IR光谱。结果与化学信息学方法的结果一致。热处理使胶态胰蛋白酶粉末失活的结果表明，结晶胰蛋白酶的纳米结构因加热而改变，这反映出β-折叠主要发生了转变，因为1634cm(-1)处的峰随着脱水而降低。FT-IR化学信息学方法能够对干燥过程中胰蛋白酶散装粉末的生物活性进行固态定量分析。
