【摘要】 用分子生物学方法表达、纯化了游仆虫中心蛋白及N-端半分子,用铽荧光探针法、离子竞争法研究了pH7.4,0.01mol·L-1Hepes条件下中心蛋白与铽、钙的结合性质。结果表明中心蛋白有4个铽结合部位,其中2个为高亲合结合部位、2个为低亲合结合部位。具有2个低亲合结合部位的中心蛋白半分子与铽结合的条件常数是(2.13±0.10)×105L·mol-1,与钙结合的条件常数是(7.52±0.02)×102L·mol-1。更多还原

【Abstract】 Euplotes centrins (EoCens) including full-length protein (apoEoCen) and semi-molecule centrin (N- apoEoCen) were expressed and purified by biological engineering. The binding of terbium (Tb3+) to apoEoCen was monitored by fluorescence in 0.01 mol·L-1 N-2-hydro-xyethylpiperazine-N-2-ethanesulfonic acid (Hepes), at pH 7.4. It could be seen that the protein binds two Tb3+ with high affinity and two Tb3+ with low affinity. The reac- tions between N-apoEoCen and Tb3+ or Ca2+ were also studied by Tb3+ fluorescence probe and ionic competition in 0.01 mol·L-1 Hepes, at pH 7.4. On the basis of fluorescence titration curves, the conditional binding constant of Tb2-N-EoCen was determined to be (2.13 ±0.10) ×105 L·mol-1 and the relative binding constant of Ca2-N-EoCen was calculated to be (7.52 ±0.02) ×102 L·mol-1.更多还原