Effects of additives on heat denaturation of rhDNase in solutions.
OBJECTIVE
To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions.


METHODS
Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T(m)) and enthalpy (H(m)) of rhDNase. The effects of denaturants (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 temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca(2+) stabilizes rhDNase against thermal denaturation and elevates T(m) and H(m) while Mg(2+), Mn(2+) and Zn(2+) destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T(m) and H(m). All destabilization of rhDNase can be prevented by adding Ca(2+) to the solutions.


CONCLUSIONS
CaCl(2) and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HCl destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca(2+) the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.
Chan
H K
HK
Department of Pharmaceutical Research and Development, Genentech, Inc. South San Francisco, California 94080, USA.
Au-Yeung
K L
KL
Gonda
I
I
D002122:Calcium Chloride / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002412:Cations; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003850:Deoxyribonuclease I / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions
D002412:Cations; D011994:Recombinant Proteins; D012996:Solutions; D003850:Deoxyribonuclease I; D002122:Calcium Chloride