Tsitologiya  2013  55 (12) : 886–892

PHYSICAL-CHEMICAL PROPERTIES OF THE MUTANT (PROTEIN) FORM OF D-GLUCOSE/D-GALACTOSE-BINDING PROTEIN GGBP/H152C WITH AN ATTACHED FLUORESCENT DYE BADAN A.V. Fonin,1,* Olga V. Stepanenko,1 O.I. Povarova,1 E.A. Volova,1,2 E.M. Philippova,1,3 G.S. Bublikov,1 I.M. Kuznetsova,1,3 A.P. Demchenko,4 K.K. Turoverov 1,3 1 Institute of Cytology RAS, St. Petersburg, 2 St. Petersburg State University, 3 St. Petersburg State Polytechnical University and 4 Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kiev; * e-mail: alexfonin@incras.ru The influence of various factors on the physico-chemical characteristics and complexation of glucose with a mutant form of D-glucose/D-galactose-binding protein which can be regarded as a sensor of the glucometer, namely the protein GGBP/H152C with solvatochromic dye BADAN attached to the cysteine residue Cys 152, has been investigated. The point mutation His152Cys and attaching BADAN reduced the affinity of the mutant form GGBP/H152C to glucose more than 8-fold compared to the wild type protein. This allows using this mutant for the determination of sugar content in biological fluids extracted by transdermal technologies. Sufficiently rapid complexation of GGBP/H152C with glucose (the time of protein-glucose complex formation is not more than three seconds even in solutions with a viscosity of 4 cP) provides timely monitoring changes in the concentration of sugar. The changes of ionic strength and pH within the physiological range of values of these variables do not have significant influence on fluorescent characteristics of GGBP/H152C-BADAN. At acidic pH some of the molecules GGBP/H152C is in the unfolded state. It has been shown that mutant form GGBP/H152C has relatively low resistance to guanidine hydrochloride denaturing effects. This result indicates the need for more stable proteins to create a sensor for glucose biosensor system. Key words:  fluorescence, biosensor, glucose, fluorescent dye |  Back   |  Contents   |  Main  |