Study On Regulative Function To Hemoglobin Structure By Surfactants And Polysaccharides

With the continuous expansion of applications of surfactants,the interactions between surfactants and proteins have been widely investigated.The study on the interaction between surfactants and proteins can help to understand the principle of surfactants as a protein denaturing agent and solvent,so as to achieve its better use in various fields.As natural biological macromolecules,the interactions between polysaccharides and proteins have great influence on the structure and functional properties of proteins,which is often used to modify the protein.The new characteristics of the complexes formed by proteins and polysaccharides have great significance to the development of new foods.Three single-chain surfactants with different hydrophobic chain length,N-dodecyl-N-?2-hydroxy ethyl?-N,N-dim ethyl ammonium bromide?DHDAB?,N-tetradecyl-N-?2-hydroxyethyl?-N,N-dimethyl ammonium bromide?THDAB?and N-cetyl-N-?2-hydroxyethyl?-N,N-dimethyl ammonium bromide?CHDAB?have been synthesized and charactered by elemental analysis,infrared spectrum and 1H NMR.Three gemini surfactants with different spacer length:1,7-heptanediaminium,N,N'-dihexadecyl-N,N,N',N'-thtramethyl-,dibromide?16-7-16?,1,8-octanediaminium,N,N'-dihexadecyl-N,N,N',N'-thtramethyl-,dibromide?16-8-16?,1,9-nonanediaminium,N,N'-dihexadecyl-N,N,N',N'-thtramethyl-,dibromide?16-9-16?have been synthesized and charactered by elemental analysis,infrared spectrum and 1H NMR,and the physical chemistry properties of six surfactants were also studied.The decomposition temperature of three single-chain and three gemini surfactants was studied by thermogravimetric analysis,and the results showed that the longer hydrophobic chain length of single-chain surfactant and longer spacer of gemini surfactant has the higher decomposition temperature.The results of conductivity experiment showed that the single-chain surfactants with longerhydrophobic chain and gemini surfactants with longer spacer have higher critical micelle concentration?CMC?,and the CMC increases with the increase of temperature.UV-visible spectroscopy,fluorescence spectroscopy,circular dichroism spectroscopy,isothermal titration microcalorimetry and Zeta potential measurement were used to study the interactions between single-chain surfactants/gemini surfactants and hemoglobin?Hb?.Conclusions are as follows:?1?The results of UV-Vis spectra showed that both the single-chain and gemini surfactants can interact with the hemoglobin to form hemichrome.At the low range of concentration,the interaction strength between surfactants and hemoglobin increases with the increase of the carbon chain length of single-chain surfactants and the spacer length of gemini surfactants.?2?The intrinsic fluorescence spectra experimental results showed that surfactants have enhancement function to the fluorescence of hemoglobin,and the fluorescence intensity increases with the increase of the carbon chain length of single-chain surfactants and gemini surfactant spacer length.pH and temperature also have significant effect on the interactions between surfactants and Hb.?3?The results of synchronous fluorescence spectra showed that the fluorescence of hemoglobin mainly comes from the tryptophan residue,and the hydrophobic chains of the surfactants are inserted into the hydrophobic cavity of hemoglobin,which make the protein structure become loose.?4?The results of CD spectra showed that surfactants make the secondary structure of hemoglobin loose,and the a-helical structure are decreased.At low concentration,the influence of the surfactant on the secondary structure of hemoglobin is CHDAB>THDAB>DHDAB,which is DHDAB>THDAB>CHDAB at high concentration.The influence of gemini surfactants on the secondary structure of hemoglobin is 16-9-16>16-7-16>16-7-16 at low concentration,which is 16-8-16>16-8-16>16-9-16 at higher concentration.The experiments indicated that the single-chain surfactants have more important effect on the structure of Hb than that of Gemini surfactants.?5?ITC and Zeta potential measurements showed that the electrostatic and hydrophobic interactions are the main driving force for the interactions between surfactants and hemoglobin.The turbidity analysis experiments,particle size determination experiments,UV Vis spectroscopy,fluorescence spectroscopy and infrared spectroscopy were used to study the interactions between carrageenan and hemoglobin,and the conclusions are as follows:?1?Through the determination of the turbidity of K-carrageenan-hemoglobin complex solution at different pH,the turbidity-pH curve is divided into four areas:1.co-soluble biopolymers;2.soluble complexes;3.insoluble complexes;4.co-soluble biopolymers.?2?The ratio and total concentration of protein and polysaccharide have important effect on the division of four regions:when immobilized the ratio of protein and polysaccharide,the higher of the total concentration of protein and polysaccharide,the more insoluble complexes are formed,and the insoluble complex region shift toward higher pH direction;When immobilized the total concentration of protein and polysaccharide,the four regions move the direction to low pH value with the increase of protein proportion,and the fourth regions in low pH?co-soluble biopolymers region?are disappeared.?3?The presence of four regions in turbidity analysis experiments was further verified by particle size measurement,and the change of hemoglobin structure was studied by UV-vis and fluorescence spectroscopy.?4?Infrared spectra of hemoglobin,carrageenan and carrageenan-hemoglobin complexes were also determined.Compared the absorption peaks of the functional groups,it can be seen that the interactions are mainly caused by electrostatic interactions between the-NH3+ of hemoglobin and the-OH,-SO₃^-of ?-carrageenan.