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The binding of carbon dioxide by horse haemoglobin
Insights
This study modified horse hemoglobin to investigate carbon dioxide binding and the Bohr effect. Results confirm hemoglobin binds CO2 via alpha-amino groups, with alpha-chain amino groups influencing the Bohr effect.
Area of Science:
- Biochemistry
- Physiology
Background:
- Hemoglobin's function involves oxygen transport and allosteric regulation.
- Understanding hemoglobin's interaction with carbon dioxide and its role in the Bohr effect is crucial for respiratory physiology.
Purpose of the Study:
- To investigate the role of specific alpha-amino groups in horse hemoglobin's carbon dioxide binding and Bohr effect.
- To characterize modified hemoglobin derivatives with targeted reactions on alpha-amino groups.
Main Methods:
- Preparation of three modified horse hemoglobin derivatives using cyanate reactions on alpha-amino groups.
- Measurement of Hill constants (n) to assess co-operative interactions.
- Analysis of charge differences at varying pH to determine protonic charge changes.
- Evaluation of the alkaline Bohr effect and carbon dioxide binding capacity.
Main Results:
- All modified hemoglobins exhibited co-operative interactions (n values between 2.0 and 2.6).
- Modified hemoglobins showed distinct charge differences from normal hemoglobin at acidic pH.
- Two derivatives (alpha(c)(2)beta(2) and alpha(c)(2)beta(c)(2)) displayed a 25% decrease in the alkaline Bohr effect.
- Carbon dioxide binding varied, with deoxy-alpha(c)(2)beta(c)(2) showing no increased binding compared to its oxy form, unlike other derivatives.
Conclusions:
- The study confirms that hemoglobin binds carbon dioxide primarily through its four terminal alpha-amino groups under physiological conditions.
- The two terminal alpha-amino groups on the alpha-chains are implicated in mediating the Bohr effect.
- Specific modifications to hemoglobin's alpha-amino groups significantly alter its CO2 binding and Bohr effect characteristics.
Abstract:
1. Three modified horse haemoglobins have been prepared: (i) alpha(c) (2)beta(c) (2), in which both the alpha-amino groups of the alpha- and beta-chains have reacted with cyanate, (ii) alpha(c) (2)beta(2), in which the alpha-amino groups of the alpha-chains have reacted with cyanate, and (iii) alpha(2)beta(c) (2), in which the two alpha-amino groups of the beta-chain have reacted with cyanate. 2. The values of n (the Hill constant) for alpha(c) (2)beta(c) (2), alpha(2)beta(c) (2) and alpha(c) (2)beta(2) were (respectively) 2.5, 2.0 and 2.6, indicating the presence of co-operative interactions between the haem groups for all derivatives. 3. In the alkaline pH range (about pH8.0) all the derivatives show the same charge as normal haemoglobin whereas in the acid pH range (about pH6.0) alpha(c) (2)beta(c) (2) differs by four protonic charges and alpha(c) (2)beta(2), alpha(2)beta(c) (2) by two protonic charges from normal haemoglobin, indicating that the expected number of ionizing groups have been removed. 4. alpha(c) (2)beta(2) and alpha(c) (2)beta(c) (2) show a 25% decrease in the alkaline Bohr effect, in contrast with alpha(2)beta(c) (2), which has the same Bohr effect as normal haemoglobin. 5. The deoxy form of alpha(c) (2)beta(c) (2) does not bind more CO(2) than the oxy form of alpha(c) (2)beta(c) (2), whereas alpha(c) (2)beta(2) and alpha(2)beta(c) (2) show intermediate binding. 6. The results reported confirm the hypothesis that, under physiological conditions, haemoglobin binds CO(2) through the four terminal alpha-amino groups and that the two terminal alpha-amino groups of alpha-chains are involved in the Bohr effect.