ISSN:
1573-904X
Keywords:
heat-induced denaturation
;
MGDF
;
reversibility
;
sucrose
;
circular dichroism
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract Purpose. The present study was performed to examine the effect of solution conditions on the reversibility of the thermal denaturation of megakaryocyte growth and development factor (rHuMGDF). Methods. Changes in the far U V CD spectra of rHuMGDF with temperature were used to monitor the thermal denaturation of the protein, and the recovery of folded protein following a return to room temperature. The effect of protein concentration, scan rate, and buffer composition on thermal denaturation and on the reversibility were determined. Surface tension measurements were used to determine the effect of this unfolding reaction on the surface adsorption of the protein. Sedimentation velocity was used to assess recovery of native monomer and the size of soluble aggregates. In addition, monomeric protein remaining in solution after incubation at 37°C for 2 weeks in either 10 mM imidazole of 10 mM phosphate was determined. Results. In phosphate buffer the rHuMGDF irreversibly precipitates upon unfolding under all the conditions examined. In imidazole the unfolding is at least partially reversible, with no visible precipitate seen; the degree of reversibility increased by lowering both protein and salt concentrations, and the amount of time spent at elevated temperature. In order to compare thermal unfolding occuring with different degrees of reversibility, the melting temperature was defined as the temperature at which melting begins. The melting temperature itself is relatively independent of the buffer composition, or experimental conditions. At low protein concentrations the protein stabilizer sucrose had a marginal effect on the thermal transition of rHuMGDF, while at protein concentrations of about 2 mg/ml the inclusion of sucrose increased the apparent melting temperature by about 4°C, to that seen at low protein concentrations, but had little effect on the reversibility of denaturation. Inclusion of 1 or 2 M urea did not affect the reaction. Surface tension measurements of rHuMGDF solutions showed little difference before and after melting, and in the presence or absence of sucrose. When unfolding is irreversible, the MGDF appears to form soluble aggregates of tetramers to 14-mers, while under reversible conditions native monomer is recovered. More monomeric MGDF remained in solution following storage for 2 weeks at 37°C in imidazole than in phosphate, in both the presence and absence of sucrose. Conclusions. These results can be explained by assuming that thermal denaturation proceeds as a two-step reaction, the first step being the equilibrium between folded and unfolded states, while the second step is a slow irreversible aggregation. The different buffer systems affect the rate of the aggregation step, but not the intrinsic thermal stability nor the rate of the unfolding step.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1018893115557
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