Structural and Functional Properties of Human Hemoglobins Reassembled after Synthesis in Escherichia coli

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• 作者：Hilda L. Hui ; Jeffrey S. Kavanaugh ; Michael L. Doyle ; Anita Wierzba ; Paul H. Rogers ; Arthur Arnone ; Jo M. Holt ; Gary K. Ackers ; and Robert W. Noble
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：January 19, 1999
• 年：1999
• 卷：38
• 期：3
• 页码：1040 - 1049
• 全文大小：162K
• 年卷期：v.38,no.3(January 19, 1999)
• ISSN：1520-4995

文摘

Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al.[(1992) Biochemistry 31, 8619-8628] has been transformed into a functionally homogeneous proteinwhose properties closely approximate those of normal hemoglobin A. Both of the mages/gifchars/alpha.gif" BORDER=0> and mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> chains of thishemoglobin contain a valine-methionine substitution at position 1 in order to accommodate the differencein specificity of the protein-processing enzymes of procaryotes. Despite extensive purification, functionalhomogeneity of the E. coli expressed hemoglobin was achieved only by the complete disassembly of thehemoglobin into its component mages/gifchars/alpha.gif" BORDER=0> and mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> globins and their reassembly in the presence of hemin. Thekinetics of CO combination and the thermodynamics of O₂ binding and cooperativity of the reassembledmages/gifchars/alpha.gif" BORDER=0>V1M-mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">V1M hemoglobin closely approximate those of HbA. The mages/gifchars/alpha.gif" BORDER=0> globin obtained from the E. coliexpressed hemoglobin was also combined with normal human mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> chains and hemin to form the mages/gifchars/alpha.gif" BORDER=0>V1Mvariant. The mages/gifchars/alpha.gif" BORDER=0>+M variant of HbA, in which the normal N-terminal valine of the mages/gifchars/alpha.gif" BORDER=0> chains is preceded bya methionine residue, was prepared by the same procedure. The kinetics of the reactions of CO with themages/gifchars/alpha.gif" BORDER=0>V1M and mages/gifchars/alpha.gif" BORDER=0>+M variants are similar to those for HbA. The equilibria of oxygen binding to mages/gifchars/alpha.gif" BORDER=0>V1M andHbA are similar whereas mages/gifchars/alpha.gif" BORDER=0>+M exhibits a significantly higher oxygen affinity. The three-dimensionalstructures of mages/gifchars/alpha.gif" BORDER=0>V1M and mages/gifchars/alpha.gif" BORDER=0>+M offer an explanation for the latter affinity difference. Although the structuresof mages/gifchars/alpha.gif" BORDER=0>V1M and HbA, which have been determined by X-ray crystallography, are virtually indistinguishableexcept at the N-terminal residues, that of mages/gifchars/alpha.gif" BORDER=0>+M indicates the displacement of a solvent molecule, possiblya chloride ion, from arginine 141mages/gifchars/alpha.gif" BORDER=0>. Such an alteration in an anion binding site could result in increasedoxygen affinity.