钝顶螺旋藻C-藻蓝蛋白分子内不同基团间能量传递的研究

STUDIES ON ENERGY TRANSFER BETWEEN DIFFERENT GROUPS IN SPIRUNINA PLATENSIS C-PHYCOCYANIN

于1996年8-12月，从钝顶螺旋藻中分离纯化C-藻蓝蛋白，并用荧光光谱法对C-藻蓝蛋白分子内不同基团间的能量传递进行研究。结果表明，C-藻蓝蛋白分子内芳香族氨基酸残基能将能量传至与脱辅基蛋白共价结合的色基，从而使色基产生相应的荧光；C-藻蓝蛋白分子240－245nm的荧光激发峰产生于二硫键，它也能将吸收的能量传至色基；同时还发现，溶液状态下的C-藻蓝蛋白分子内，色氨酸残基可能位于分子内部的疏水区，而二硫键则位于分子表面的非荷电区域。这些均可作为开展C-藻蓝蛋白分子天然构象研究的结构表征。

From August to December of l996, C-phycocyanin was purified from Spirulina platensis,and energy transfer between different groups in C-phycocyanin was studied in terms of its fluorescence spectra. The purification of the C-phycocyanin was based upon the use of a hydroxylapatite chromatography and the purity was 5 (A620 /A280). The C-phycocyanin showed two fluorescence peaks at 355 nm and 65Onm at the excitation wavelength of 28Onm, which is attributed to the absorption of aromatic amino acid residues in apoprotein of the C-phycocyanin; this is also the case for the C-phycocyanin when excited at 24Onm. If the C-phycocyanin was denatured, then the fluofescence peak at 65Onm decreased greatly, whilst the fluorescence peak at 355nm was shifted to 38Onm and the relative fluorescence intensity increased. However, if the C-phycocyanin was denatured or DTT was added to the C-phycocyanin solution, then all fluorescence peaks disappeared at the excitation wavelength of 24Onm. The results show that the energy which is absorbed by the aromatic amino acid residues can be transfered from the aromatic amino acid residues to the chromophore which is conjugated to apoprotein in C-phycocyanin in solution to make it produce fluorescence, it is found that the fluorescence excitation peak at 24O-245nm results from disulfide bonds in C-phycocyanin and the energy can also be transfered from disulfide bonds to chromophore of C-phycocyanin. With the help of some quenchers, the location of the tryptophan residues of the C-phycocyanin was found in the hydrophobic region of the inner molecules and that of disulfide bonds in the nonelectrostatic part of the C-phycocyanin molecule surface. The mechanism of energy transfer between the aromatic amino acid residues in apoprotein of C-phycocyanin and the chromophore of C-phycocyanin may be associated with the Forster dipole-dipole resonance because of the overlap of fluorescence emission peak of tryptophan residues at 355nm with the absorption peak of the chromophores of C-phycocyanin, which are called phycocyanobilin, at 360nm. On the other hand, the mechanism of energy transfer between the disulfide bonds of the apoprotein of C-phycocyanin and the chromophore of C-phycocyanin may be related to excitation coupling because the disulfide bonds in apoprotein do not produce fluorescence.Thus, the energy transfer between different groups can serve as structure characters inphycobi1iproteins.