Pathway and Mechanism of pH Dependent Human Hemoglobin Tetramer-Dimer-Monomer Dissociations

Abstract

Hemoglobin dissociation is of great interest in protein process and clinical medicine as well as in artificial blood research. However, the pathway and mechanisms of pH-dependent human Hb dissociation are not clear, whether Hb would really dissociate into monomers is still a question. Therefore, we have conducted a multi-technique investigation on the structure and function of human Hb versus pH. Here we demonstrate that tetramer hemoglobin can easily dissociate into dimer in abnormal pH and the tetramer → dimer dissociation is reversible if pH returns to normal physiological value. When the environmental pH becomes more acidic (<6.5) or alkaline (>8.0), Hb can further dissociate from dimer to monomer. The proportion of monomers increases while the fraction of dimers decreases as pH declines from 6.2 to 5.4. The dimer → monomer dissociation is accompanied with series changes of protein structure thus it is an irreversible process. The structural changes in the dissociated Hbs result in some loss of their functions. Both the Hb dimer and monomer cannot adequately carry and release oxygen to the tissues in circulation. These findings provide a comprehensive understanding on the pH-dependent protein transitions of human Hb, give guideline to explain complex protein processes and the means to control protein dissociation or re-association reaction. They are also of practical value in clinical medicine, blood preservation and blood substitute development.

Estimation of protein concentration at high sensitivity using SDS‐capillary gel electrophoresis‐laser induced fluorescence detection with 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde protein labeling

“…Note that although alpha and beta globin are listed separately, they were contained within one sample of hemoglobin. MS measurement confirmed that under conditions used hemoglobin was dissociated into monomers, in agreement with literature , and it was considered as an equimolar mixture of alpha and beta globins.…”

Conformational Changes and Competitive Adsorption between Serum Albumin and Hemoglobin on Bioceramic Substrates

“…Consequently, we have to interpret the low number of interacting spin pairs to be a consequence of protein dissociation on the BC surface. A similar dissociation has been observed at extreme pH values (below 6.5 and above 8.0), or for low sample concentrations (<100 μ m ) …”

“…This means that dissociation happens either at the α 1 β 2 /α 2 β 1 or at the α 1 β 1 /α 2 β 2 sliding interfaces. The α and β subunits interact along the α 1 β 1 (α 2 β 2 ) packing interface by forming tight contacts involving approximately 34 residues, whereas the α 1 β 2 (α 2 β 1 ) sliding interface involves only about 19 residues . The formation of α 1 β 1 and α 2 β 2 is therefore more likely .…”

“…As imilar dissociation has been observed at extreme pH values (below 6.5 and above 8.0), or for low sample concentrations (< 100 mm). [42] Pulse EPR distance measurementsa lso provide information about the number of interacting spins, which can furtherb e used for structure determination. The modulation depth of the background-corrected DEER time-domaind ata is directly related to the number of interacting spins per protein molecule, [33] Figure 5.…”

“…The a and b subunits interact along the a 1 b 1 (a 2 b 2 )p acking interface by forming tight contacts involving approximately 34 residues,w hereas the a 1 b 2 (a 2 b 1 )s liding interface involves only about 19 residues. [42] The formation of a 1 b 1 and a 2 b 2 is therefore more likely. [42,43] We tried to reduce the number of dissociated hemoglobin dimers by using an organic layer as ab uffer between the substrate and the proteins.F or this, the BC substrate was first coatedw ith unlabeled BSA molecules and then dried at room temperature for 24 hp rior to hemoglobina dsorption.…”