TY - JOUR
T1 - A flow procedure to determine oxygen binding isotherms for low affinity and easily oxidized hemoglobins
AU - Astatke, Mekbib
AU - McGee, William A.
AU - Parkhurst, Lawrence J.
N1 - Funding Information:
The determination of binding constants for hemoglobins has been an important problem in physical biochemistry for the past century (Bert, 1878; Hfifner, 1890; Bohr, 1904; Hill, 1910; Adair, 1925; Lyster et aL, 1951; Imai, 1982). Among the various hemoglobins, those from certain fish and reptiles are unusual in that they show a remarkable change in affinity with pH (Root, 1931; Noble et al., 1970), a phenomenon known as the "Root effect". In carp hemoglobin, this has been shown (Tan et al., 1972) to correspond to a switching of the hemoglobin from an "R" like state at high pH to a T state at low pH, in which cooperativity is essentially lost at the extremes of pH. The transition to the T state in carp appears complete even at pH 6 if the allosteric effector IHP is present in solution. This result has allowed carp Hb to serve as a model hemoglobin for studying allosteric processes (Henry et al., 1985; Parkhurst et al., 1983). Unfortunately, for many hemoglobins and myoglobins, as the affinity is lowered, there is a corresponding increase in the rate of autoxidation (Wallace et al., 1982; Springer et al., 1989), considerably complicating the problem of determining binding constants. The situation is further complicated by the possibility of heme heterogeneity in oxygen binding in the T state, since, at least in human Hb, it is the beta chain to which IHP binds (Arnone *This work was supported by grant NIH DK36288. /'Author to whom correspondence should be addressed. Abbreviations--Standard abbreviations of Hb, Hb +, HbO2 and HbCN denote deoxy, met, oxy and eyano-met forms of hemoglobin; the half-saturation oxygen activity is denoted by ~, and the Hill number at ,~' by n*. Other abbreviations are: DTT, dithiothreitol; IHP, inositol hexaphosphate.
PY - 1992/4
Y1 - 1992/4
N2 - 1. 1. The determination of binding isotherms for low affinity hemoglobins is particularly difficult because of rapid oxidation. 2. 2. In carp Hb (pH 6 + 1HP, 25°C), one-quarter of the hemes are oxidized within 3 min, preventing the accurate determination of even P50 or X. 3. 3. We circumvent this problem by rapidly flowing HbO2, initially at pH 9 (X = 1.4 μM), against a low pH buffer to bring the system rapidly to equilibrium in the low affinity form. Diode-array spectrophotometry allows a complete spectrum to be obtained < 5 sec, after flow ceases, before significant oxidation has occurred. In tandem with the stopped flow apparatus is an oxygen electrode to measure O2 activity. 4. 4. At 22°C, the half-saturation oxygen activity (X) is 227 μM, and the Hill number is 0.91, for carp Hb (T state) implying significant differences in the O2 affinities of the alpha and beta chain hemes or, two different T states.
AB - 1. 1. The determination of binding isotherms for low affinity hemoglobins is particularly difficult because of rapid oxidation. 2. 2. In carp Hb (pH 6 + 1HP, 25°C), one-quarter of the hemes are oxidized within 3 min, preventing the accurate determination of even P50 or X. 3. 3. We circumvent this problem by rapidly flowing HbO2, initially at pH 9 (X = 1.4 μM), against a low pH buffer to bring the system rapidly to equilibrium in the low affinity form. Diode-array spectrophotometry allows a complete spectrum to be obtained < 5 sec, after flow ceases, before significant oxidation has occurred. In tandem with the stopped flow apparatus is an oxygen electrode to measure O2 activity. 4. 4. At 22°C, the half-saturation oxygen activity (X) is 227 μM, and the Hill number is 0.91, for carp Hb (T state) implying significant differences in the O2 affinities of the alpha and beta chain hemes or, two different T states.
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U2 - 10.1016/0305-0491(92)90359-Y
DO - 10.1016/0305-0491(92)90359-Y
M3 - Article
C2 - 1611887
AN - SCOPUS:0026590227
VL - 101
SP - 683
EP - 688
JO - Comparative Biochemistry and Physiology -- Part B: Biochemistry and
JF - Comparative Biochemistry and Physiology -- Part B: Biochemistry and
SN - 0305-0491
IS - 4
ER -