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Submitted to: Journal of the American Oil Chemists' Society
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 5/26/2014 Publication Date: 8/20/2014 Citation: He, Z., Uchimiya, M., Cao, H. 2014. Intrinsic fluorescence excitation-emission matrix spectral features of cottonseed protein fractions and the effects of denaturants. Journal of the American Oil Chemists' Society. 91(9):1489–1497. Interpretive Summary: Cottonseed protein has received increased interests for food and non-food industrial applications. However, fundamental knowledge on cottonseed protein is needed to better understand the functional and physicochemical properties of cottonseed protein, and to promote its industrial use. Therefore, in this work, we investigated the intrinsic fluorescence excitation-emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, and the effects of denaturants urea, guanidine hydrochloride and sodium dodecyl sulfate on these cottonseed protein preparations. The EEM spectra showed two contour peaks. Addition of denaturants gradually upshifted the emission maxima of both peaks for CSPI and CSPa. The emission maxima of CSPw were unchanged by denaturation. These observations indicated that the tryptophan residues (fluorescence source) in the native CSPI and CSPa were protected within the micro hydrophobic environment, and gradually become water accessible with progressing denaturation. However, the fluorescence intensity of Peak 1 of all three protein samples decreased with increasing denaturant concentrations, suggesting similarity in some conformational changes in the three samples. Further exploration of the fluorescence mechanism of Peak 1 is needed to understand such similar conformational changes. Technical Abstract: To better understand the functional and physicochemical properties of cottonseed protein, we investigated the intrinsic fluorescence excitation-emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, and the effects of denaturants urea, guanidine hydrochloride (GdmCl) and sodium dodecyl sulfate (SDS). The EEM showed two contour peaks with the excitation wavelengths at 226 nm (Peak 1) and 277 nm (Peak 2); emission wavelength was 335 nm for CSPI and CSPa, and 353 nm for CSPw. Peak 2 was similar to the single emission spectral maximum of seed proteins observed with fixed excitations at 260 and 295 nm reported in literature. Little information on Peak 1 was available in literature. Addition of denaturants gradually shifted the emission maxima of both peaks to around 353 nm for CSPI and CSPa. The emission maxima (353 nm) of CSPw were unchanged by denaturation. These observations indicated that the tryptophan residues (fluorescence source) in the native CSPI and CSPa were protected within the micro hydrophobic environment, and gradually become water accessible with progressing denaturation. On the other hand, the tryptophan residues in native CSPw were already in contact with water. However, the fluorescence intensity of Peak 1 of all three protein samples decreased with increasing denaturant concentrations, suggesting similarity in some conformational changes in the three samples. Further exploration of the fluorescence mechanism of Peak 1 is needed to understand such similar conformational changes. |
