Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.

Area of Science:

• Biochemistry
• Biophysics
• Protein dynamics

Background:

• Asymmetric hemoglobin (Hb) hybrids, composed of deoxy and liganded dimers, have been studied for their stability.
• Previous research suggested enhanced stability for doubly liganded hybrids, with dissociation times over 100 seconds.

Purpose of the Study:

• To investigate the stability and dissociation kinetics of asymmetric hemoglobin hybrids.
• To determine the lifetime of the [deoxy/azido-met] hybrid (species 21) and assess its stability relative to symmetric counterparts.

Main Methods:

• Utilized double mixing stopped-flow experiments to create and study asymmetric Hb hybrids.
• Generated [deoxy dimer/azido-met dimer] hybrids within 200 ms by mixing dithionite with oxyHb and azido-metHb solutions.
• Probed allosteric states by CO binding after variable aging times of the asymmetric hybrids.

Main Results:

• Freshly formed asymmetric hybrids exhibited rapid CO binding, characteristic of the R-state Hb.
• Aging of hybrids from 0.1 to 10 seconds increased the fraction of slow CO binding, indicating dissociation into deoxy Hb tetramers.
• Simulated tetramer to dimer dissociation rates for species 21 were approximately 1.5-1.9/second, similar to liganded Hb, not deoxy Hb.

Conclusions:

• The asymmetric [deoxy/azido-met] hybrid (species 21) has a short lifetime of about one second.
• These findings do not support the symmetry rule, which predicts enhanced stability for asymmetric Hb hybrids.
• The observed kinetics are inconsistent with a slow R to T transition, suggesting rapid dimer dissociation is the primary pathway.