We found that recombinant human adult hemoglobin (rHb A) expressed in
Escherichia colishowed heterogeneity of components with the intensity of a positive CD b
and 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 ![](/images/gifchars/beta2.gif)
subunits, with contents of ~50
and >80% in SP-2
and SP-3, respectively.Rotation of the heme 180
![](/images/entities/deg.gif)
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 b
and in both the near-UV
and Soret regions, but rHbA with reversed heme exhibits a very weak positive CD b
and at 260 nm
and a prominent negative CDb
and 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.