Positive and Negative Cooperativities at Subsequent Steps of Oxygenation Regulate the Allosteric Behavior of Multistate Sebacylhemoglobin

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• 作者：Enrico Bucci ; Anna Razynska ; Herman Kwansa ; Zygmunt Gryczynski ; John H. Collins ; Clara Fronticelli ; Ron Unger ; Michael Braxenthaler ; John Moult ; Xinhua Ji ; and Gary Gilliland
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：March 19, 1996
• 年：1996
• 卷：35
• 期：11
• 页码：3418 - 3425
• 全文大小：425K
• 年卷期：v.35,no.11(March 19, 1996)
• ISSN：1520-4995

文摘

Cross-linked human hemoglobin (HbA) is obtained by reaction withbis(3,5-dibromosalicyl)sebacate. Peptide maps and crystallographic analyses confirm thepresence of the 10 carbon atom longsebacyl residue cross-linking the two beta2.gif" BORDER=0 ALIGN="middle">82 lysines of the beta2.gif" BORDER=0 ALIGN="middle">-cleft(DecHb). The Adair's constants, obtainedfrom the oxygen binding isotherms, show that at the first step ofoxygenation normal hemoglobin andDecHb have a very similar oxygen affinity. In DecHb negativebinding cooperativity is present at thesecond step of oxygenation, which has an affinity 27 times lower thanat the first step. Positive cooperativityis present at the third binding step, whose affinity is 380 times thatof the second step. The fourth bindingstep shows a weak negative cooperativity with an affinity one-half thatof the third step. Crystals ofdeoxy-DecHb diffracted to 1.9 Å resolution. The resulting atomiccoordinates are very similar to thoseof Fermi et al. [(1984) J. Mol.Biol.175, 159-174] and Fronticelli et al. [(1994) J.Biol. Chem. 269,23965-23969] for deoxy-HbA. The electron density map ofdeoxy-DecHb indicates the presence of the10 carbon bridge between the beta2.gif" BORDER=0 ALIGN="middle">82 lysines. Molecular modelingconfirms that insertion of the linker intothe T structure requires only slight displacement of the two beta2.gif" BORDER=0 ALIGN="middle">82lysines. Instead, insertion of the linkerinto the R and R2 structures [Shaanan (1983) J. Mol. Biol.171, 31-59; Silva et al. (1992) J. Biol.Chem.267, 17248-17256] is hindered by serious stericalrestrictions. The linker primarily affects thepartiallyand fully liganded states of hemoglobin. The data suggest in DecHbconcerted conformational changesat each step of oxygenation.