Other
Scientific paper
Jun 1998
adsabs.harvard.edu/cgi-bin/nph-data_query?bibcode=1998aipc..430..332b&link_type=abstract
The eleventh international conference on fourier transform spectroscopy. AIP Conference Proceedings, Volume 430, pp. 332-335 (1
Other
Infrared And Raman Spectroscopy
Scientific paper
The three-dimensional structure of many proteins and complex polypeptides is critically sensitive to the environment to which such a biopolymer is subjected. For example, in some cases lyophilization, which entails a complete cycle of freezing, drying in vacuo, and warming to ambient, brings about unpredictable changes to the secondary structure (conformation). Various research and development groups have suggested that the addition of sucrose or other oligosaccharides to protein solutions prior to lyophilization works to maintain the structural integrity of the native macromolecule. Less information is available, however, with regard to whether or not the presence of sugar inhibits thermal denaturation of proteins in solution. In the present study, FTIR spectroscopic examination of solutions of the monoclonal antibody in D2O suggests that 6% (w/V) added sucrose produces little change in the observed mean temperature of thermal denaturation, Tm~68 °C. Yet, when the protein solution is held at constant temperature in the range between about 60 and 75 °C, the presence of sucrose significantly alters the rate at which the protein unfolds. Based on the disappearance of the strong band near 1634 cm-1 associated with intramolecular β-structure of the native protein, the unfolding process was found to follow first-order kinetics. In the absence of sugar, the slope of a plot of ln (intensity) versus time yielded a first order rate constant of kl=2.65 hr-1 at 69 °C. By contrast, when the protein solution contained 6% sucrose (w/V), kl=1.85 hr-1 at the same temperature. Measuring the rate at different temperatures around Tm indicates that protein solutions containing sucrose unfold more slowly above ~68 °C than does the protein without the excipient. At lower temperatures, however, the opposite is true; the presence of the sugar enhances the rate of denaturation. Arrhenius plots of ln kl versus 1/T indicates that Ea=~600 kJ/mol for the protein alone, but only ~330 kJ/mol with sucrose present.
Baldoni J. M.
Byler D. M.
Dal Monte P. R.
Lee David L.
Sokoloski T. D.
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