Detection and characterization of hemoglobin dissociation and aggregation using microcalorimetry

Abstract

Using micro-differential scanning calorimetry (μDSC) to probe hemoglobin structural changes, previously unreported large exothermic events have been repeatedly detected at temperatures below the tertiary-to-secondary structural transition temperature (ca. 72 °C), which could be important in understanding protein subunit association. Given the importance of protein–protein interactions and protein solution stability, this event was characterized with respect to concentration and scan rate. This detailed analysis revealed the formation of aggregates that differed in size, appearance, and settling time. While repeat μDSC scans confirmed that this aggregation event was irreversible when samples were heated beyond 55 °C, it was also found that aggregation could be prevented through the addition 0.02 M urea. All evidence points to a significant occurrence of a kinetic pathway that leads to an intermediate aggregation event that is believed to be formed from dissociated hemoglobin subunits.