Postnikova G B, Yumakova E M
Laboratory of Biophysics of Redox Proteins, Institute of Biological Physics of the USSR Academy of Sciences, Moscow Region.
Eur J Biochem. 1991 May 23;198(1):241-6. doi: 10.1111/j.1432-1033.1991.tb16007.x.
The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2-13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5-10.8 but change significantly at pH less than 5.5 and pH greater than 10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH less than 4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio I325/I350 which diminishes at pH greater than 9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the 'salt bridges' essential for the maintenance of the native Mb structure in the N-terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.
对与原卟啉IX复合的抹香鲸脱辅基肌红蛋白的卟啉和色氨酸荧光在pH值2至13范围内进行了研究。结果表明，结合到血红素裂隙中的原卟啉的荧光和吸收光谱在pH值5.5至10.8范围内保持不变，但在pH值小于5.5和大于10.8时会发生显著变化，这分别是由于复合物的酸变性和碱变性以及原卟啉IX的解离所致。在相同的pH范围内，由于作为猝灭剂的原卟啉从复合物中去除，色氨酸荧光的量子产率急剧增加。色氨酸荧光的其他参数（最大位置、半高宽和光谱形状）在碱性区域也会发生变化。在酸性pH范围内，这些参数仅在pH值小于4.3时发生变化，这表明色氨酸周围环境比血红素裂隙区域对变性更稳定。在pH值5.5至10.9之间，脱辅基肌红蛋白与原卟啉IX的复合物处于天然状态，色氨酸荧光的主要参数保持不变，除了I325/I350比值在pH值大于9.5时减小。其变化先于复合物的碱变性，并且可以通过N端区域中维持天然肌红蛋白结构所必需的“盐桥”断裂引起的局部构象变化来解释。从脱辅基肌红蛋白、肌红蛋白以及原卟啉IX与脱辅基肌红蛋白之间的复合物获得的荧光数据能够使人比较它们的结构，并评估卟啉大环和铁原子在天然肌红蛋白结构形成及其功能发挥中的作用。
