Tuning the Properties of Hb Intercalated in the Galleries of α-ZrP with Ionic Strength:  Improved Structure Retention and Enhanced Activity

The influence of ionic strength on the behavior of met-hemoglobin (Hb) bound to layered α-zirconium phosphate (α-ZrP) was investigated. Small increases in the ionic strength (5 mM to 80 mM K₂HPO₄, pH 7.2) indicated large improvements in the bound protein structure, activity, and stability. The number of ZrP units occupied per bound Hb (stoichiometry) increased from 773 to 2000 units. The Soret absorption band and the corresponding circular dichroism (CD) spectra (Soret) indicated that there are considerable improvements in the protein structure around the heme pocket with increase in ionic strength. Monitoring the UV−CD in the 190−250 nm range indicated only minor changes with ionic strength, and some formation of α-helical coiled coils was indicated. The peroxidase-like activity of the bound Hb increased approximately threefold when the ionic strength was increased from 10 mM to 40 mM K₂HPO₄ (pH 7.2). Free Hb did not indicate similar improvements in structure or activity. Differential scanning calorimetric studies showed that bound Hb was denatured over a wide range of temperature and a significant portion of the protein was stabilized by the solid. This study provides a simple method to improve the bound protein properties, and such approaches aid in engineering more effective synthetic materials to maximize bound enzyme function.