Monday, May 5, 2008
9-39

Effect of polyethylene glycol on the stability of green fluorescent protein

Letícia C. L. Novaes1, Flavio M. Magaldi1, Hans-Olof Johansson2, Adalberto Pessoa Jr.1, and Thereza C. V. Penna1. (1) Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580 Bl 16, São Paulo, 05508-900, Brazil, (2) Lund University, Lund, Sweden

Polymers can affect the thermostability of proteins. In some studies melting temperature (Tm) of lysozyme was lowered upon addition of polyethylene glycol 1,000g/mol at pH from 3.0 to 3.8. On the other hand, there are examples showing an opposite effect i.e. an increased stability of the protein caused by the presence of PEG. There is a large interest in using polymers to stabilize protein solution for pharmaceutical applications. We investigated the effect of PEG 10,000g/mol on the thermostability of GFP at 80ºC. From an entropic point of view the presence of a polymer favours the native structure of a protein through the confinement of available space. However, polymers may also interact hydrophobically and favour protein denaturation. The concentration of GFP was determined by spectrofluorometry, adjusted for the excitation and emission wavelengths (λex=394nm,λem=509nm). GFP was denatured by heating the solution in a quartz cuvette in pH 7,70. The value D of GFP (7,87mg/mL) at 80ºC in the absence of PEG 10,000 was 39.29min. The value D decreased to 26.38min after increasing the concentration of PEG10,000 to 5% (w/w) of PEG concentration. For other concentrations of PEG, the values of D were: 16.89min for 10% (w/w); 12.34min for 15% (w/w); 12.62min for 20% (w/w); 9.55min for 25% (w/w); 8.48min for 30% (w/w); 8.09min for 35% (w/w); and 9.24min for 40% (w/w). These results show that the PEG 10,000 did not protect the native structure of GFP, and it favoured the denaturation of GFP at 80ºC.

Acknowledgements: CNPq, CAPES and FAPESP.


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