荧光探针Bis-ANS和磷酸丙糖异构酶的相互作用

THE INTERACTION OF FLUORESCENCE PROBE BIS-ANS WITH TRIOSE PHOSPHATE ISOMERASE

研究了极性荧光探针Bis-ANS和磷酸丙糖异构酶的相互作用。我们发现由磷酸丙糖异构酶(TIM)中Trp残基和结合在TIM分子上的Bis-ANS之间的能量传递引起的Trp残基荧光的淬灭呈双相性,表明Bis-ANS在TIM分子上可能有2个不相同的结合位点,其结合的解离平衡常数Kd分别为3.3μM和17.0μM。底物GDP引起已结合的Bis-ANS荧光强度进一步增强和荧光谱的蓝移说明GDP可影响Bis-ANS在TIM分子上结合部位的构象,使其疏水性增强。我们还观察到由于结合在同一TIM分子上的Bis-ANS之间的能量传递引起的退偏振,进一步证明Bis-ANS有2个结合部位在1—2800bar压力范围里,增高压力引起结合在TIM分子上的Bis-ANS荧光进一步增强和光谱蓝移,说明TIM在压力下解离成亚基的过程中发生了Weber提出的"conformationaldrift。

The interaction between the fluorescence polar probe bis- (8-anilinonaph-thalene-1-sulfonate) (bis-ANS) and triosephosphate isomerase from yeast (TIM) was described in this paper. The biphasic quenching of the TIM intrinsic fluorescence caused by the radia tionless energy transfer from tryptophane residues in TIM to bonnd bis-ANS, indicated the presence of two types of bis-ANS binding site in TIM molecule. The estimated dissociation constant, Kd , are 3.3 μM and 17.0 μM respectively. The presence of TIM substrate D-gly-ceraldehyde-3 phosphate (GDP) causes the increase in the fluorescence intensity of bound bis-ANS, which implies that GDP can effect the conformation of the bis-ANS binding site in TIM. The dependence of depolarization of bound bis-ANS on the bis-ANS concentration in solution indicates that the energy transfer between the Lound bis-ANSs in TIM occurs. The increase in fluorescence intensity and the blue shift of fluorescence spectrum of bound bis-ANS caused by high hydrostatic pressure indicate that the conformational drift takes place during the dissociation of TIM into monomer.