Effect of Reversed Heme Orientation on Circular Dichroism and Cooperative Oxygen Binding of Human Adult Hemoglobin
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- 作者：Masako Nagai ; Yukifumi Nagai ; Yayoi Aki ; Kiyohiro Imai ; Yoshinao Wada ; Shigenori Nagatomo ; Yasuhiko Yamamoto
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：January 15, 2008
- 年：2008
- 卷：47
- 期：2
- 页码：517 - 525
- 全文大小：214K
- 年卷期：v.47,no.2(January 15, 2008)
- ISSN：1520-4995
文摘
We found that recombinant human adult hemoglobin (rHb A) expressed in Escherichia colishowed heterogeneity of components with the intensity of a positive CD band at 260 nm and that it couldbe resolved into three components (SP-1, SP-2, and SP-3) by SP-Sepharose column chromatography. 1HNMR revealed that SP-1 is identical with native Hb A, while SP-2 and SP-3 largely contain the reversedheme isomer in both the 
and 
subunits, with contents of ~50 and >80% in SP-2 and SP-3, respectively.Rotation of the heme 180
about the 5,15-meso axis (reversed heme) causes an interexchange of themethyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examinethe effect of the modification of the heme-protein contact on the structure and function of Hb A, wecompared the 1H NMR, CD, and oxygen binding properties of the three components with those of nativeHb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHbA with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CDband in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allostericeffector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A withreversed heme. These results indicate that changes in the heme-globin contact exert a discernible influenceon CD spectra and cooperative oxygen binding.
and subunits, with contents of ~50 and >80% in SP-2 and SP-3, respectively.Rotation of the heme 180 about the 5,15-meso axis (reversed heme) causes an interexchange of themethyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examinethe effect of the modification of the heme-protein contact on the structure and function of Hb A, wecompared the 1H NMR, CD, and oxygen binding properties of the three components with those of nativeHb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHbA with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CDband in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allostericeffector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A withreversed heme. These results indicate that changes in the heme-globin contact exert a discernible influenceon CD spectra and cooperative oxygen binding.