Barber M J, Trimboli A J, McIntire W S
Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa.
Arch Biochem Biophys. 1993 May 15;303(1):22-6. doi: 10.1006/abbi.1993.1250.
The low molecular weight "blue" copper protein, azurin, has been purified from Pseudomonas putida (NCIB 9869) to homogeneity using various chromatographic techniques including reverse-phase HPLC. The amino acid sequence of the N-terminus of the reduced and carboxymethylated protein yielded a single sequence corresponding to AECKV. The complete sequence, comprising 128 amino acid residues with a C-terminal sequence corresponding to TVTLK, was determined from the peptides obtained from a Staphylococcus aureus V8 digest of the protein and confirmed using peptides obtained following cyanogen bromide and endoprotease Asp-N digests. The amino acid sequence contained three cysteine residues at positions 3, 26, and 112, was devoid of tryptophan, and showed closest similarity (90% identical residues) to the previously determined sequence of azurin isolated from Pseudomonas fluorescens biotype B [Ambler, R.P. (1971) in Developpements Recents Dans L'Etude Chimique De La Structures Des proteins (Preverio, A., Pechere, J.-F., and Coletti-Preverio, M.-A., Eds.), pp. 289-305, INSERM, Paris]. Examination of the complete sequence indicated P. putida azurin contained unique Asp and Ala residues at positions 19 and 21, respectively, that have not been found in any other azurin sequence.
利用包括反相高效液相色谱在内的多种色谱技术，从恶臭假单胞菌（NCIB 9869）中纯化出了低分子量的“蓝色”铜蛋白——天青蛋白，使其达到了同质状态。还原和羧甲基化蛋白N端的氨基酸序列产生了一个对应于AEKV的单一序列。完整序列由128个氨基酸残基组成，其C端序列对应于TVTLK，该序列是从该蛋白的金黄色葡萄球菌V8消化产物中获得的肽段确定的，并通过溴化氰和内蛋白酶Asp-N消化后获得的肽段进行了确认。氨基酸序列在第3、26和112位含有三个半胱氨酸残基，不含色氨酸，与先前确定的从荧光假单胞菌生物型B中分离出的天青蛋白序列显示出最高的相似性（90%的相同残基）[安布勒，R.P.（1971年），载于《蛋白质结构化学研究的最新进展》（普雷维里奥，A.、佩舍尔，J.-F.和科莱蒂-普雷维里奥，M.-A.编），第289 - 305页，法国国家卫生与医学研究所，巴黎]。对完整序列的检查表明，恶臭假单胞菌天青蛋白在第19和21位分别含有独特的天冬氨酸和丙氨酸残基，这些残基在任何其他天青蛋白序列中均未发现。
