| Object:Human serum albumin(HSA)and immunoglobulin G(IgG)are proteins that exert important physiological and biochemical functions in the body.Its structure is susceptible to a va-riety of factors such as temperature,pH,salt ion concentration,etc.,ultimately altering the activity of the protein.It is important to explore an analytical tool that characterizes structural changes in HSA and IgG.Near-infrared spectroscopy and mid-infrared spectroscopymainly reflect the funda-mental frequency,frequency doubling,combined frequency and combined frequency absorption of molecular vibration,and is a powerful tool for studying protein structure.However,due to the reso-lution and characteristics of the infrared spectrum,its application in protein structure analysis is li-mited.The emergence of chemometrics technology has brought new hopes to solve the above prob-lems.Therefore,this study mainly uses infrared spectroscopy to investigate the effects of tempera-ture,pH and salt ion concentration on the structural changes of HSA and IgG by using wavelet transform and two-dimensional correlation spectroscopy,In order to provide a means of characteri-zation of protein structure and function,and provide a theoretical basis for the optimization of HSA and IgG production process conditions.Methods:In this experiment,two kinds of proteins,HSA and IgG,were selected as the re-search objects.Near-infrared spectra of two proteins at different temperatures and different NaCl concentrations were collected by near-infrared spectroscopy,and the obtained spectra were processed and analyzed.The two-dimensional/attenuated full-emission infrared spectroscopy analy-sis technique was used to study the secondary structure changes of HSA under different pH buffer systems,and the protein change process was explained according to the order of changes in absorp-tion peaks.Results:In this paper,near-infrared spectroscopy was used to study the structural changes of HSA and IgG under temperature changes.The microscopic molecular structure changes were ex-plored from the near-infrared spectroscopy.The temperature rise changed the content of water mo-lecules in different structures.The structure of the protein was changed.In aqueous solution,the structure of 60 mg/mL HSA changed significantly at around 60 ?;the 5 mg/mL IgG aqueous solu-tion changed structurally at around 61 ?,and precipitated at 65 ?.It is indicated that near-infrared spectroscopy can characterize the subtle structural changes of proteins,and can provide some help for tracking the changes of protein activity in the production process.The near-infrared spectrosco-py technique was used to investigate the structural changes of HSA and IgG in different concentra-tions of NaCl aqueous solution.The change of salt ion concentration,that is,the change of solution ionic strength,can destroy the hydrophobic interaction and change the molecular conformation of the protein.As the concentration of NaCl increases,Na+,C1-,and some water molecules combine to form a coordination compound,thereby destroying the hydrogen bond of the water molecule.The above structural changes can be reflected in the near-infrared spectrum of the sample,which lays a foundation for exploring the mechanism of NaCl influence on protein structure.The two-dimensional/attenuated full-emission infrared spectroscopy technique was used to study the second-ary structure changes of HSA under different pH buffer systems.It is known from the analysis that at least four carboxyl groups with different hydrogen bonding degrees participate in the structural change.The process,in different pH ranges(pH 7.2-5.0,pH 5.2-4.4,pH 4.6-3.8,pH 3.8-3.0),all change from the carboxyl structure,so it is speculated that in the acidic environment,the carboxyl group Protonation of the mass may be an important cause of changes in the structure of the induced protein.Conclusion:The effect of temperature and NaCl concentration on the structure of HSA and IgG proteins was elucidated by this study,and the effect of pH on HSA structure was elucidated.The increase of temperature changes the content of water molecules in different structures and changes the structure of the protein;the increase of NaCl concentration will destroy the hydrogen bond of water molecules;the protonation of carboxyl groups in acidic environment may induce the change of protein structure.The important reason is to lay a theoretical foundation for the actual production process of protein. |
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