Protein Polymer Conjugates: Improving the Stability of Hemoglobin with Poly(acrylic acid)

The synthesis, characterization, and evaluation of a novel polymer-protein conjugate are reported here. The covalent conjugation of high-molecular weight poly(acrylic acid) (PAA) to the lysine amino groups of met-hemoglobin (Hb) resulted in the covalent conjugation of Hb to PAA (Hb-PAA conjugate), as confirmed by dialysis and electrophoresis studies. The retention of native-like structure of Hb in Hb-PAA was established from Soret absorption, circular dichroism studies, and the redox activity of the iron center in Hb-PAA. The peroxidase-like activities of the Hb-PAA conjugate further confirmed the retention of Hb structure and biological activity. Thermal denaturation of the conjugate was investigated by differential scanning calorimetry and steam sterilization studies. The Hb-PAA conjugate indicated an improved denaturation temperature (T(d)) when compared to that of the unmodified Hb. One astonishing observation was that polymer conjugation significantly enhanced the Hb-PAA storage stability at room temperature. After 120 h of storage at room temperature in phosphate-buffered saline (PBS) at pH 7.4, for example, Hb-PAA retained 90% of its initial activity and unmodified Hb retained <60% of its original activity under identical conditions of buffer, pH, and temperature. Our conjugate demonstrates the key role of polymers in enhancing Hb stability via a very simple, efficient, general route. Water-swollen, lightly cross-linked, stable Hb-polymer nanogels of 100-200 nm were produced quickly and economically by this approach for a wide variety of applications.