EFFECTS OF ADDITIVES ON HEAT DENATURATION OF RHDNASE IN SOLUTIONS

Purpose. To study the thermal stability of recombinant human deoxyribo nuclease I (rhDNase) in aqueous solutions. Methods. Differential scann ing calorimetry (DSC) was used to measure the denaturation or melting temperature (T-m) and enthalpy (H-m) of rhDNase. The effects of denatu rants (guanidine HCl and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6-7. Results. The T-m and H -m of rhDNase in pure water were measured as 67.4 degrees C and 18.0 J /g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperatur e, indicating that the thermal denaturation was irreversible. The latt er was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that duri ng heat denaturation, rhDNase formed soluble high molecular weight agg regates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca2+ stabilizes rhDNase against thermal denaturation and elevates T-m and H-m while Mg2+, Mn2 and Zn2+ destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T-m and H-m. All des tabilization of rhDNase can be prevented by adding Ca2+ to the solutio ns. Conclusions. CaCl2 and sugars were found to stabilize rhDNase agai nst thermal denaturation while divalent cations, urea and guanidine HC l destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca2+ the protective effect is believed to be due t o an ordering of the rhDNase structure in its native state, and by pre vention of breaking of a disulfide bridge, thus making it less suscept ible to unfold under thermal stress.