Free dye affinity partitioning of glucose-6-phosphate dehydrogenase and hexokinase in aquaeoud two-phase systems

• Grant number: 00/01010-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: February 01, 2001
• End date: January 31, 2002
• Field of knowledge: Agronomical Sciences - Food Science and Technology - Food Engineering
• Principal Investigator: Adalberto Pessoa Junior
• Grantee: Xu Yan
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The applications of aqueous two-phase systems to problems of separation and extraction of macromolecules, organelles, and cells have increased markedly, and their use has been extended into most areas of biology. Common aqueous two-phase system is not selective enough to provide the extreme purity usually desired, so the affinity partitioning was developed. PEG polymer modified by grafting reactive dyes to the terminal oxygen atoms have been utilized in affinity-based separation to increase the selectivity of certain biomolecules for PEG-rich phase in aqueous two-phase systems. For simplifying, free dye affinity partitioning technique was developed. In this study, Cibacron blue F3GA, Procion Red HE-3B, and Procion Yellow HE-3G will be empioyed as free affinity ligands in PEG/hydroxypropyl starch two-phase system for the selective partition of G6PDH and HK. To strip the enzymes from the affinity phase, the PEG/sulfate and PEG/phosphate system are empioyed. Through the screening process, the proper system and optimum operation conditions will be determined. The scale-up of the process to 1 liter will be performed to verify the feasibility of the method. Through the investigation, a high selectivity, large capacity, low cost and simple purification technique for the glucose-6-phosphate dehydrogenase and hexokinase is to be anticipated. (AU)