Study On The Intermolecular Interaction Of Human Serum Albumin During Ethanol Precipitation Based On Aquaphotomics

Human serum albumin(HSA)is the earliest blood product extracted from human plasma,produced on a large scale and used in clinical applications.As the most abundant protein in plasma,HSA plays an important physiological role in the human body.Due to the limitation of the raw material plasma,the situation of "short supply" often occurs.At present,the cold ethanol precipitation method is mainly used in the production of HSA in the world,and the production of HSA is usually controlled by the fixed process parameters.Due to the individual differences,there are differences in the plasma content and composition from different donors.The use of fixed process parameters can easily cause the waste of raw material.Therefore,in-depth research on the ethanol precipitation mechanism of HSA and applying "quality by design" strategy for different sources of plasma to improve the yield and quality of products,have become one of the most important aspects for scientific researchers.The ethanol precipitation is the core process in HSA separation and purification.However,the ethanol precipitation mechanism of HSA is not completely clear until now,which limits the understanding of the physiological functions of HSA and obtaining high quality products.The aggregation and precipitation of HSA is affected by many factors,and its separation and purification are also mainly based on the dynamic equilibrium between dissolution and precipitation caused by different physical and chemical properties of plasma proteins under different production parameters.HSA has a large molecular weight and complex structure,and the intermolecular interactions in HSA have a significant effect on the precipitation process.Therefore,it is one of the important basic work for HSA precipitation to study the thermodynamic properties of protein solutions,especially the intermolecular interactions and the solution structure.Based on the above,the study on intermolecular interaction of HSA in precipitation process was carried out,including the structure and hydration behavior of HSA induced by ethanol concentration and different pH.As an important molecular spectroscopic technique,infrared spectroscopy has certain application and research foundations in the field of blood products.Because of its sensitivity to weak hydrogen bonding,it is widely used to study the hydrogen bonding,hydration,and self-association behavior of various compounds and become one of the important spectral analysis techniques in biological research.By using spectroscopy technique,aquaphotomics takes water as the research object to reflect the interaction between proteins and solvents at molecular level.Therefore,infrared aquaphotomics was mainly used to study the intermolecular interaction of HSA in ethanol precipitation process,including the study on the intermolecular interaction of ethanol-water solution,the study on the hydration behavior of HSA aqueous solution with different concentration and the study on the structure and hydration behavior of HSA induced by ethanol and different pH.The specific chapters are as follows:1 Study on the molecular interaction in ethanol-water mixturesIn this chapter,near infrared(NIR)spectroscopy combined with chemometrics was used to detail the mechanism of the molecular interaction in ethanol-water binary mixtures.The ethanol solution in range of 0%?10%(v/v)was investigated with excess spectra and Gaussian fitting with the knowledge of aquaphotomics.It was indicated that water can be a probe for sensing molecular interactions and when the concentration of ethanol is lower than 5%,the main form of the intermolecular interaction in the mixtures is hydrophobic hydration,while the ethanol concentration higher than 5%,the O-H from water and ethanol form the fixed interaction by hydrogen bonds.Meanwhile,it was found that the hydrogen bonding strength between ethanol and water reach the maximum at 40%concentration.This may be the reason of many abnormal properties in the mixtures and cause the end of precipitation of plasma proteins.The two-dimensional correlation analysis implies that in 10%?40%solution,ethanol tends to interact with water molecules firstly,but with the further increasing concentration,the self-association between ethanol molecules has the priority,which trigger a weakening of the interaction between water and ethanol and disintegration of water and ethanol cluster.In conclusion,the findings provide important theoretical support for the changes in the hydrogen bonding network in ethanol-water solution.As an important solvent for HSA precipitation,the research of ethanol-water solution is of great practical significance for the separation and purification of plasma proteins.If a thoroughly understanding of the hydration behavior of different plasma proteins in the ethanol-water binary system was obtained at the molecular level,it will be beneficial to obtain high-quality plasma protein products and maximize the use of limited plasma resources.2 Analysis of the hydration water around human serum albuminThe perturbation of the water structure due to the presence of HSA was investigated by using NIR spectroscopy.Calculation of the change of molar extinction coefficients ??for the water in the mixtures provides an evidence for the nonlinear behavior of the HSA aqueous solutions by looking at the modifications induced in water spectra.The hydration number calculated by McCabe-Fisher method with density data suggests a more complex relationship between the surface of HSA and water.The phenomenon of decreasing in hydration number with increasing HSA concentration was explained by using an overlapping hydration layer model and hydrogen bond network.Analysis of the resolved hydration spectra for HSA confirmed that free water participates in the formation of hydration layers.The transition between different water species demonstrated that the hydration layer is dynamic and fluctuating affected strongly by the protein molecules.In high concentration HSA solution,the "strengthen" water hydrogen bonds at the protein interface form the less-defective hydrogen-bond network by inhibiting the fragmentation of water-water hydrogen bonds.When HSA concentration was higher than 10 mg/mL,the water species with 3 and 4 hydrogen bonds reached equilibrium.The findings provide important theoretical support during HSA purification which is of vital practical significance to obtain high quality HSA preparations.Meanwhile,this study provides a new method and perspectives for the study of protein hydration behavior.3 pH induced the secondary structural changes and hydration variation of HSAMiddle-infrared(MIR)spectra was applied to study the pH effect on the secondary structure of HSA in buffer solution.It was found that the secondary structure of HSA changed significantly around pH 4.5,that is,the transition from N form to F form by using moving window two-dimensional correlation analysis.Two-dimensional correlation analysis was used to analyze the N form,N-F form,and F form of HSA solution separately.It was found that the protonation of the carboxyls in side chains of HSA may be an important reason for the secondary structure change.At the same time,by using NIR differential spectrum analysis,it was found that protein hydration experienced a sharp decay around pH 4.5,which means that hydrated water participated in the secondary structure change of HSA.The two-dimensional correlation analysis was used,and found that during the conformational changes,different hydrogen-bonded water species participate in the change of HSA secondary structure.This study explores the impact of the acidic pH environment experienced in production on the structure and hydration of HSA,and provides theoretical guidance for pH adjustment4 Study on the ethanol-dependent the secondary structural changes and hydration behaviors of HSAThe influence of ethanol on the secondary structure of HSA in aqueous solution was investigated using two-dimensional correlation MIR spectroscopy.It was found that low concentrations of ethanol have the effect of enhancing the a-helical structure,and also promoting the formation of intermolecular ?-sheet structures which cause the precipitation of protein.These changes may be achieved through the changes of carboxyl group in side chains.The results of the apparent molar volume show that the addition of ethanol competes with the hydration water on the surface of HSA,leading to the weakened hydration,resulting in aggregation and precipitation of HSA.The NIR spectra were collected for hydration analysis.The hydration spectrum of HSA was analyzed with McCabe-Fisher method.Following the concept of aquaphotomics,the hydration spectra of HSA was analyzed.It was observed that the conformation of the hydrated water molecules of HSA changed differently with different ethanol concentration.Water species with different hydrogen bonds participate in the process of HSA precipitation process,which can be further used as markers in the precipitation.This study deepens the understanding of the ethanol precipitation mechanism and provides theoretical support for process optimization.5 Study on the ethanol-dependent the secondary structural changes and hydration behaviors of HSA at isoelectric pointTwo-dimensional correlation MIR spectroscopy was used to investigate the effect of ethanol on the secondary structure of HSA in buffer solution at around isoelectric point pH.At the isoelectric point pH,ethanol mainly promotes the formation of intermolecular?-sheet structures,that is,it mainly promotes the precipitation of proteins.Low concentrations of ethanol can enhance the a-helical structure of HSA.The calculation of the apparent molar volume of HSA and the analysis of the NIR difference spectrum show that the isoelectric point pH and ethanol have a synergistic effect on the dehydration process of HSA.The McCabe-Fisher method was used to analyze the hydration spectrum of HSA,and the water structure of the hydration layer was analyzed following the concept of aquaphotomics,showing that different water species participate in the precipitation process of HSA.The study laid the foundation for revealing the mechanism of ethanol precipitation.To sum up,a preliminary study on the structure and hydration changes of HSA induced by pH and ethanol was carried out.Firstly,the ethanol-water solution and HSA aqueous solution were studied,the results show that strong hydrogen bonding is formed between ethanol molecules and water molecules at the concentration of about 40%,which is the main reason for the dehydration of protein surfaces.The hydration and the water structure changes in hydration layer in different concentration of HSA aqueous solution were analyzed,showing a non-linear relationship with HSA concentration.It provides a reference for the selection of HSA concentration in the ethanol precipitation.Secondly,the effects of ethanol and the effect of pH on the secondary structure and hydration of HSA were studied.The results show at acidic pH,too low a pH can cause the secondary structural change of HSA,which may begin with the protonation of the carboxyl groups of the side chains.Hydration water participates in the secondary structural change of HSA.The addition of low-concentration ethanol enhances the a-helical structure of HSA.At the same time,ethanol can promote the formation of intermolecular ?-sheet structures,weakened the hydration of HSA,and eventually lead to the occurrence of HSA precipitation.Finally,the effects of ethanol and isoelectric pH on the secondary structure and hydration of HSA were studied.The results show that ethanol and isoelectric pH have a synergistic effect,which accelerates the dehydration process of HSA.From the perspective of molecular spectroscopy,this study clarified the key intermolecular interactions of the HSA in ethanol precipitation process.It deepens the understanding of the structural change characteristics and hydration behavior of the HSA in the ethanol precipitation process,and lays the foundation for a complete explanation of the mechanism in HSA precipitation.At the same time,this study provides a certain theoretical basis for the selection of process parameters in plasma protein precipitation and provides key scientific support for optimizing production processes and obtaining high-quality plasma protein products.Meanwhile,the essence and general law of the action mechanism in the similar system are obtained via the study of weak interaction of HSA in different solution,which is the theoretical basis for further research on complex biological systems.