三维荧光技术在肌氨酸氧化酶活性蛋白检测中的应用

Application of three-dimensional fluorescence technique in the detection of sarcosine oxidase of active protein

利用三维荧光检测肌氨酸氧化酶(SOX)内源荧光物质的特征峰,探究SOX热失活机理以及在粗酶液中快速检测活性蛋白的应用。采集色氨酸(Trp)、酪氨酸(Tyr)、黄素腺嘌呤二核苷酸(FAD)和SOX三维荧光光谱数据,确定SOX中Trp、Tyr和FAD内源荧光峰位置。考察不同温度(30℃~90℃)热处理15 min后SOX内源荧光峰位置和强度变化,建立FAD荧光强度与SOX活性蛋白浓度之间的线性关系模型,在重组菌粗酶液中验证方法可行性。结果表明:SOX中主要存在氨基酸残基和FAD两种内源荧光特征峰。光电信增管(PMT)电压400 V下检测氨基酸荧光峰,在热处理过程中,随着温度升高,氨基酸荧光峰波长范围基本不变,荧光强度从592.75 a. u.增加到1104 a. u.。PMT电压700 V下检测FAD荧光峰,30℃~55℃热处理后SOX样品中FAD荧光峰波长范围不变,荧光强度从890.16 a. u.下降到804.21 a. u.;60℃~90℃时,FAD荧光峰变为游离态FAD特征峰,荧光强度从115.72 a. u.增加到142.13 a. u.,高温使FAD由结合态转为游离态,导致酶蛋白基本失活。根据SOX中FAD特异性荧光峰和荧光强度可预测粗酶液中SOX活性蛋白浓度。室温下,FAD荧光强度与SOX活性蛋白质量浓度在0.05~1.0 mg/mL范围内线性关系良好,拟合曲线R^(2)=0.9929。经验证,曲线预测均方根误差(RM SEP)为0.0978,平均浓度回收率为99.7%。通过检测SOX特异性的内源FAD三维荧光,不仅能解析SOX的热失活机理,还能根据FAD荧光强度快速测量粗酶液中SOX蛋白浓度,为其他以FAD为辅酶的黄素类蛋白快速检测分析提供借鉴。

Three-dimensional fluorescence spectroscopy was used to detect the characteristic peaks of the endogenous fluorescence of sarcosine oxidase( SOX),to investigate the mechanism of thermal inactivation of SOX and its application in the rapid detection of active proteins in crude enzyme solutions. Three-dimensional fluorescence spectroscopy of tryptophan( Trp),tyrosine( Tyr),flavin adenine dinucleotide( FAD),and SOX were collected to determine the locations of the endogenous fluorescence peaks of Trp,Tyr,and FAD in SOX.The change of SOX endogenous fluorescence peak position and intensity after heat treatment at different temperatures( 30℃-90℃) for 15 minutes was investigated,and a linear relationship model between FAD fluorescence intensity and SOX active protein concentration was established to verify the feasibility of the method in the crude enzyme solution of recombinant bacterial. The results indicated that two endogenous fluorescence characteristic peaks,amino acid residues and FAD,were mainly present in SOX. The amino acid fluorescence peak was detected at a PMT voltage of 400 V. During the heat treatment,the wavelength range of the amino acids fluorescence peak was basically constant with increasing temperature,and the fluorescence intensity increased from 592. 75 a. u. to 1104 a. u.. The wavelength range of the FAD fluorescence peak detected in the SOX sample at a PMT voltage of 700 V after heat treatment at 30℃-55℃ remained unchanged,and the fluorescence intensity decreased from 890. 16 a. u. to 804. 21 a. u.,at 60℃-90℃,the FAD fluorescence peak changed to the free state FAD characteristic peak,and the fluorescence intensity increased from 115. 72 a. u. to142. 13 a. u.. The high temperature changes the FAD from the bound state to the free state,resulting in the basic inactivation of the enzyme protein. The linear relationship between FAD fluorescence intensity and SOX active protein concentration was good in the range of 0. 05-1. 0 mg/m L at room temperature,with R;of 0. 9929 for the fitted curve. The RMSEP was verified to be 0. 0978 and the mean concentration recovery was 99. 7%.The detection of three-dimensional fluorescence spectroscopy of FAD in SOX can not only analyze the mechanism of thermal inactivation of SOX but also provide the rapid measurement of the protein concentration of SOX contained in the crude enzyme solution based on FAD fluorescence intensity,which provides a reference for the rapid detection and analysis of other flavin-like proteins with FAD as a coenzyme.