The artificial alpha1beta1-contact mutant hemoglobin, Hb Phe-35beta, shows only small functional abnormalities

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Hemoglobin (Hb) Philly, characterized by a Phe to Tyr mutation at position 35(C1)beta, was previously reported to exhibit non-cooperative oxygen binding, high affinity, and instability causing hemolysis.
• The absence of a specific 1H-NMR signal at 13.1 ppm in Hb Philly, compared to normal hemoglobin A (Hb A), led to its assignment to a hydrogen bond involving Tyr-35(C1)beta.

Purpose of the Study:

• To investigate the functional and structural properties of an artificial hemoglobin mutant with the same mutation as Hb Philly.
• To re-evaluate the previously reported characteristics of Hb Philly and the assignment of the 13.1 ppm NMR signal.

Main Methods:

• Construction and characterization of an artificial hemoglobin mutant.
• Assessment of oxygen binding affinity and cooperativity.
• 1H-NMR spectroscopy to analyze structural features and hydrogen bonding.

Main Results:

• The artificial mutant displayed slightly decreased oxygen affinity but near-normal cooperativity.
• The characteristic 1H-NMR signal at 13.1 ppm was present in the artificial mutant.
• No signs of instability or hemolysis were observed in the artificial mutant.

Conclusions:

• The findings suggest that the mutation at position 35(C1)beta may not lead to the severe functional and stability defects previously attributed to Hb Philly.
• The assignment of the 13.1 ppm NMR signal to a hydrogen bond involving Tyr-35(C1)beta requires further investigation.
• This study highlights the need for re-examination of the original Hb Philly characterization and associated NMR signal interpretation.