Effects of reduction and ligation of heme iron on the thermal stability of heme-hemopexin complexes.
Effects of reduction and ligation of heme iron on the thermal stability of heme-hemopexin complexes.
复制标题
血红素铁的还原和连接对血红素-血红素复合物热稳定性的影响。
DOI：
10.1023/a:1011033625009
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Morgan,WT
中科院分区：
文献类型：
--
作者：
Shipulina,NV;Smith,A;Morgan,WT
Hemopexin有两个同源结构域(N-末端和C-末端结构域)，在低自旋双组氨酸复合体中以高亲和力(Kd<；1 PM)与每摩尔1摩尔的血红素结合，并作为血红素的转运体。转运是通过内吞作用完成的，而不是蛋白质的降解。研究了影响血红素配位络合物的稳定性和体内潜在的血红素释放的因素。用吸光度和圆二色谱(CD)研究了温度对Hopexin、其N-末端结构域以及它们各自的铁、铁和钴-铁-血红素络合物的影响。根据二阶导数吸收光谱的监测，在50 mM磷酸盐缓冲液中脱氢血凝素的高阶结构以52°C的温度展开，并被150 mM的氯化钠(Tm63°C)稳定。用Soret吸光度观察到，双组氨酸与血红素的配位作用因铁血红素(Tm55.5°C)还原为铁血红素形式(Tm48°C)而大大减弱，而氯化钠通过10-15°C与铁血红素-血红素结合恢复了络合物的稳定性(Tm67°C)。冷却后，展开的脱铁血红素和脱铁血红素广泛折叠，并恢复了大量的血红素结合活性，但天然蛋白(UV区)和血红素复合体(Soret区)的特征椭圆度没有恢复，表明发生了改变的折叠形式。PH从7.4降低到6.5对脱脂蛋白的稳定性影响不大，但使血红素复合体的Tmof增加了5-12℃。脱脂蛋白N-末端结构域(Tm53℃)的稳定性与完整的血球蛋白相似，N-末端结构域的铁、铁和钴-铁-血红素复合体的Tm值分别为53℃、33℃和75℃。
Hemopexin has two homologous domains (N- and C-terminal domains), binds 1 mole of heme per mole with high affinity (Kd< 1 pM) in a low-spin bis-histidyl complex, and acts as a transporter for the heme. Transport is accomplished via endocytosis without degradation of the protein. Factors that affect stability of the heme coordination complex and potentially heme release in vivo were examined. The effects of temperature on hemopexin, its N-terminal domain, and their respective ferri-, ferro-, and CO-ferro-heme complexes were studied using absorbance and circular dichroism (CD) spectroscopy. As monitored with second-derivative absorbance spectra, the higher order structure of apo-hemopexin unfolds with a Tmof 52°C in 50 mM sodium phosphate buffer and is stabilized by 150 mM NaCl (Tm63°C). Bis-histidyl heme coordination by hemopexin, observed by Soret absorbance, is substantially weakened by reduction of ferri-heme-hemopexin (Tm55.5°C) to the ferro-heme form (Tm48°C), and NaCl stabilizes both complexes by 10-15°C. CO binding to ferro-heme-hemopexin restores complex stability (Tm67°C). Upon cooling, unfolded apo- and ferri-heme-hemopexin extensively refold and recover substantial heme-binding activity, but the characteristic ellipticity of the native protein (UV region) and heme complex (Soret region) are not regained, indicating that altered refolded forms are produced. Lowering the pH from 7.4 to 6.5 has little effect on the stability of the apo-protein but increases the Tmof heme complexes by 5-12°C. The stability of the apo-N-terminal domain (Tm53°C) is similar to that of intact hemopexin, and the ferri-, ferro-, and CO-ferro-heme complexes of the N-terminal domain have Tmvalues of 53°C, 33°C, and 75°C, respectively.