Ultra-stable hemoglobin–poly(acrylic acid) conjugates

Abstract

Stabilization of proteins against thermal deactivation is a major challenge, and a simple, facile, novel chemical approach is described here to overcome this hurdle. We report here, for the first time, the successful synthesis of ultrastable protein nanoparticles consisting of met-hemoglobin (Hb) conjugated with low molecular weight polyacrylic acid (PAA, M_w 8000) to form discrete nanoparticles. Hb–PAA nanoparticles were not deactivated when subjected to prolonged thermal treatment such as steam sterilization conditions (122 °C, 40 minutes, 17–20 psi), while the unprotected Hb lost most of its activity when subjected to the same heating conditions. Several Hb–PAA derivatives which resist thermal inactivation, in a similar manner, are produced and characterized. Interestingly, the highest activity retention, after the above thermal treatment, was ∼100% for the untreated samples. This resistance to heat is attributed to the enhanced thermodynamic stability of the Hb–PAA conjugate and improved re-folding of the denatured state to the native form, facilitated by PAA conjugation to Hb. This is a unique approach to stabilize Hb against thermal inactivation, and it is a major breakthrough in the production of stable Hb-based nanomaterials that can be safely sterilized in an autoclave for biomedical/in vivo applications.