大熊猫肌红蛋白的纯化及一级结构的研究

Studies on Purification and Primary Structure of Myoglobin from Giant Panda Skeletal Muscle

本文从大熊猫骨骼肌分离纯化了肌红蛋白,鉴定了纯度,测定了分子量及氨基酸组成。确定了该蛋白的N端及C端氨基酸。采用CNBr裂解的方法,用HPLC分离得到4个肽段,分别测定了大熊猫肌红蛋白1—52位,56—77位及132—153位氨基酸的排列顺序。大熊猫肌红蛋白共含153个氨基酸,本文已完成了96个氨基酸的序列测定。用此结果与食肉目有关动物已知肌红蛋白的氨基酸顺序进行比较,获得了有意义的结论。

Myoglobin was separated and purified from giant panda skeletal muscle by the ammonium sulfate precipitating, Sephadex G-100 and DEAE-Sepharose CL-6B chroma-tography. The homogenity of the purified myoglobin is proved by the single band in the SDS-PAGE and its molecular weight is 17800. The amino acid composition is determined. N-terminal protein sequence is determined by the ABI 470A protein sequencer and C-terminal sequence F-L-G-COOH is carried out by carboxypeptidase A method. Four CNBr cleavage fragments of myoglobin are purified by HPLC TSK SW-3000H gel filtration and C-18 RP-HPMC. The N-terminal sequence containing 52 amino acid residues is: G-L-S-D-G-E-W-Q-L-V-L-N-V-W-G-K-V-E-A-D-L-A-G-H-G-Q-E-V-L-I-R-L-F-K-G-H-P-E-T-L-E-K-F-D-K-F-K-H-L-K-S-E. The sequence of P2 pep-tide is (58-77), K-G-S-E-D-L-K-K-H-G-N-T-V-E-T-A-L-E-G-I-L-K. The sequence of p4 peptide is (132-151): K-K-A-L-E-L-F-R-N-D-I-A-A-K-Y-K-E-L-G-F. Myoglobin from giant panda skeletal muscle contains 153 amino acid residues. 96 of them were determined. Comparision with known myoglobin from other Carnivora animals was made.