Spectroscopic Studies On The Structural Changes Of Bovine Serum Albumin Induced By Environmental Variation

Proteins are the fundamental substances that make up all kinds of life in nature.In recent years,generalized two-dimensional correlation spectroscopy has been widely used to study protein structure,based on the two major advantages claimed for this technique,enhancement of spectral resolution and detection of sequential order of events by the so-called "sequential order" rules.Nonetheless,the theoretical modeling and experimental results from our group have manifested that the "sequential order" rule of this technique is not universally applicable.Besides,the new bands resolved by generalized two-dimensional correlation spectroscopy are uncertain and controversial.In this work,the structural changes of bovine serum albumin induced by environmental variation were studied by in-situ spectral analyses and generalized two-dimensional correlation spectroscopy,to explore the real structural change process of BSA,and at the same time,to have a critical analysis on the two major advantages of generalized two-dimensional correlation.The main contents are as follows:1 Secondary structure transitions of Bovine Serum Albumin(BSA)in H2O and D2O under temperature variation were investigated by in-situ IR spectral analysis and they were found to exhibit the same tendency with the only difference that the significant transition temperature of BSA's secondary structure moved towards a lower temperature in D2O.Before this significant transition temperature,the native secondary structures of BSA were essentially changing in a cooperative fashion with temperature variation,except for the intermolecular ?-sheet(high-wavenumber)which increased as a result of thermal denaturation at a later stage.After the transition temperature,the relative contents of a-helix and extended chain continued to drop sharply and they were transformed into random coil,?-tura and ?-sheet.2 2D correlation spectroscopy and in-situ fluorescence spectroscopy were used to analyze the two tryptophan residues in bovine serum albumin(BSA)under different concentration of paeonol and prulifloxacin quencher.The new features resolved by two-dimensional correlation spectroscopy may be originated from band splitting.According to the "sequential order" rules,when the quencher is paeonol,the intensity variations of Tyr is before two tryptophan residues and the intensity variations of Trp-134 is after Trp-212.Furthermore,prulifloxacin was used to quench the BSA,the intensity variations of amino acid residues is as follows:Tyr>Trp-134>Trp-212.However,in situ fluorescence spectroscopic analysis showed that the intensities of Tyr,Trp-212 and Trp-134 changed in a cooperative way,without sequential order.Therefore,the information provided by 2D correlation analysis may be not real.The experimental results indicated that the smaller paeonol has greater access to the Trp-134 in hydrophobic cavum and it can quench both Trp-134 and Trp-212 with a similar rate,but prulifloxacin has bigger volume,which is hard to enter the hydrophobic cavum,so it is easier for prulifloxacin to quench the Trp-212 on the surface.3 The time-dependent changes in IR spectra in the amide ?,? and ?' region of bovine serum albumin(BSA)induced by the hydrogen-deuterium exchange was studied by two-dimensional(2D)correlation spectroscopy and in-situ spectroscopy.In contrast with the results from Second Derivative(SD)and Fourier self-deconvolution(FSD)analyses,new features were resolved by two-dimensional correlation spectroscopy,but some of the new bands resolved may originate from band splitting,peak shift,bandwidth change,etc.The integrated sequential order of the for sub-band intensity changes obtained from two-ditnensional correlation spectroscopy is as follows:1680>1580>1660>1602 cm-1,which were contradictory with the experimental observation(1633?1655>1605?1680 cm-1.In the process of hydrogen-deuterium substitution,the secondary structural changes and side chains changes of BSA are not only a cooperative process,but also a process with local sequential order.The curve-fitting results have revealed the H/D exchanges proceed in the extended chain with weak hydrogen bonding and the most abundant a-helix structure in BSA.However,the amide protons in the ?-sheet structures are resistant to the H/D exchange in 120 minutes.