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| C. Ciências Biológicas - 10. Microbiologia - 2. Microbiologia Aplicada |
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| STABILITY OF GREEN FLUORESCENT PROTEIN (GFPUV) IN CHLORINE SOLUTIONS OF VARYING PH |
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| Elaine Chau 1, Marina Ishii 1, Thereza Christina Vessoni Penna 1 e Olivia Cholewa 2 |
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| 1- Depto de Tecnologia Bioquímico-farmacêutica, Faculdade de Ciências Farmacêuticas - USP
2- Molecular Probes, Inc., Eugene, OR, USA 97402. Email: olivia.cholewa@probes.com
, UNIVERSIDADE DE SÃO PAULO - USP |
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| Green fluorescent protein (GFPuv) is an excellent biological indicator (BI) due to its ability to be easily monitored in a wide variety of applications. The purpose of this work was to study GFPuv stability in chlorinated water for injection (WFI) and chlorinated potassium phosphate buffer (10 mM; pH 6 and 7), with and without agitation, to determine (i) the exposure time required for the chlorine and pH to lower fluorescence intensity 90% (decimal reduction time, D-value, min, 25oC), and (ii) to determine which buffer system provides optimal conditions to analyze the effectiveness of chlorine upon protein denaturation, which may be related to disinfection efficacy.
Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFPuv (8.0-9.0 mg/mL) to constantly stirred solutions with chlorine concentrations ranging from: (i) 40ppm-160ppm in WFI (pH=10.6-10.9), in which the initial loss of 20-68% fluorescence occured; (ii) 10ppm-200ppm in phosphate buffers (pH=6.0-7.0), in which no change of initial fluorescence was detected up to 50ppm chlorine. The stability of GFPuv drastically dropped for chlorine concentrations >100ppm, which coincided with lowest D-values, between 1.3 min (147.11ppm) and 1.7 min (183.9ppm). For concentrations ≤100ppm chlorine, the highest GFPuv stability was exhibited in phosphate pH=7.0, with D-values ranging from 555.6 min (51.8 ppm) to 83.33 min (93.56 ppm), decreasing 10-fold to 55.6 min with 110.33ppm chlorine. For not stirred solutions, a recovery of GFPuv fluorescence was observed with chlorine at 30ppm-100ppm in WFI. Complete mixing of GFPuv in solution is essential and provides for more uniform contact of the protein to the solvent system throughout the assay and permits more accurate determination of D-values. GFPuv performed as a suitable fluorescent BI for monitoring disinfection effectiveness.
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Instituição de fomento: FAPESP, CNPq e Capes
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Trabalho de Iniciação Científica
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| Palavras-chave:
GFPuv; cloro; estabilidade |
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Anais da 57ª Reunião Anual da SBPC - Fortaleza, CE - Julho/2005
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