Protein annealing: Thermal treatment of met-hemoglobin bound to α-zirconium phosphate/phosphonates results in initial denaturation followed by recovery of activity and structure

Thermal stability of met-hemoglobin (Hb) intercalated in three layered solid materials was investigated at 60 °C. At this temperature, free Hb undergoes rapid thermal denaturation, which is accompanied by the loss of its peroxidase-like activity and protein secondary structure. Heating of Hb intercalated in the galleries of α-zirconium(IV) phosphate (at 60 °C) also indicated initial loss of peroxidase-like activity of bound Hb, within 2 h. Continued equilibration of Hb bound to this solid (at 60 °C) indicated steady increase in its activity (>50%), as a function of continued equilibration at this temperature. The extent of this recovery also depended on the type surface functions present on the solid, and these are in the order α-Zr(PO3CH2CH2COOH)2 > α-Zr(PO3CH2COOH)2 > α-Zr(HPO4)2. In the case of α-Zr(PO3CH2CH2COOH)2, the recovered activity was nearly twice as much as the activity, before heating. The circular dichroism spectra of the samples, heated at 60 °C for various time intervals, also indicated the initial loss of structure followed by significant recovery. This behavior is highly unusual, and these are the very first examples of activity recovery of proteins bound to solids, upon thermal treatment at moderate temperatures. These studies clearly indicate the important role of layered solids and their surface functions in the recovery of Hb structure. Annealing could be a useful method in recovering or improving the activities of specific proteins bound to selected solid surfaces.