| Three-dimensional fluorescence spectroscopy was a technique for characterizing intermolecular dynamics and obtaining spectral information about proteins from fluorescent molecules in biological samples.This technique didn't require complex sample pretreatment,was rapid,sensitive,and had a high application value.In this study,a flavin adenine dinucleotide(FAD)coenzyme-based sarcosine oxidase(SOX,Gen Bank:AY626822.2)obtained from previous work in the laboratory was used as the target.Using three-dimensional fluorescence spectroscopy,we investigated the fluorescence peaks of the main endogenous fluorescent substances tryptophan(Trp),tyrosine(Tyr),and flavin adenine dinucleotide(FAD)in this enzyme,and spectroscopically characterized their relationship with the concentration of SOX protein,to provide a new reference for the rapid detection and analysis in the purification system of flavin protein isolation.(1)Analysis of the three-dimensional fluorescence characteristics of the main endogenous fluorescent substances in SOX.Comparing the differences in the three-dimensional fluorescence characteristic peaks of Trp,Tyr,and FAD in the free state with those in the SOX pure enzyme.Determine the wavelength range for the detection of FAD in SOX pure enzyme at PMT 700 V:?ex=350-430 nm/?em=410-540 nm;Detection of endogenous amino acid(Trp and Tyr)fluorescence characteristic peaks in SOX pure enzyme at PMT 400 V:?ex=220-310 nm/?em=275-390 nm.The wavelength range and fluorescence intensity of SOX endogenous fluorescence peaks after heat treatment at different temperatures(30?-90?)for 15 min were studied,and it was found that the endogenous amino acid fluorescence peaks in SOX were found to be formed by amino acid residues on the surface of SOX,and the characteristic fluorescence peaks of FAD were formed by the coenzyme FAD.(2)Probing the relationship between three-dimensional fluorescence data and SOX protein concentration of SOX coenzyme FAD.FAD was a specific coenzyme in SOX.To investigate whether the presence of SOX in the crude enzyme solution can be quickly determined based on the characteristic three-dimensional fluorescence peak of FAD.The relationship between the fluorescence intensity of FAD and the concentration of SOX protein in the crude enzyme solution was also investigated.The results indicated that the linear relationship between the FAD fluorescence intensity and the concentration of SOX protein in the crude enzyme solution was good in the range of 0.05-1.00 mg·m L-1.The RMSEP was verified to be 0.0978 and the mean concentration recovery was 99.7%.(3)Analysis of the changes in the three-dimensional fluorescence characteristics of SOX endogenous amino acids(Trp and Tyr)during the affinity chromatography process.During the affinity chromatography of SOX,imidazole and hetero protein interfered with the three-dimensional fluorescence peaks of endogenous amino acids of SOX protein,and their overlapping peaks were split into three independent and non-interfering fluorescence components by using the parallel factor analysis technique(PARAFAC).Evaluation of SOX protein concentration in each eluate sample during affinity chromatography based on a fitted curve of SOX endogenous amino acid fluorescence intensity versus SOX protein concentration.The results indicated that the linearity of SOX protein concentration was good in the range of 0.05-2.50 mg·m L-1,the RMSEP was verified to be 0.0919 and the mean concentration recovery was 102.3%.The feasibility of using three-dimensional fluorescence spectroscopy to measure SOX protein concentration during affinity purification was confirmed,providing a reference for the rapid detection and analysis of other flavin-like proteins with FAD as a coenzyme. |
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