Spectral characteristics of the mutant form GGBP/H152C of D-glucose/D-galactose-binding protein labeled with fluorescent dye BADAN: influence of external factors

Alexander V Fonin; Olga V Stepanenko; Olga I Povarova; Catherine A Volova; Elizaveta M Philippova; Grigory S Bublikov; Irina M Kuznetsova; Alexander P Demchenko; Konstantin K Turoverov

doi:10.7717/peerj.275

Spectral characteristics of the mutant form GGBP/H152C of D-glucose/D-galactose-binding protein labeled with fluorescent dye BADAN: influence of external factors

PeerJ. 2014 Mar 18:2:e275. doi: 10.7717/peerj.275. eCollection 2014.

Authors

Alexander V Fonin¹, Olga V Stepanenko¹, Olga I Povarova¹, Catherine A Volova², Elizaveta M Philippova³, Grigory S Bublikov¹, Irina M Kuznetsova⁴, Alexander P Demchenko⁵, Konstantin K Turoverov⁴

Affiliations

¹ Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Science , St. Petersburg , Russia.
² Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Science , St. Petersburg , Russia ; Department of Biology, St. Petersburg State University , St. Petersburg , Russia.
³ Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Science , St. Petersburg , Russia ; Department of Physical Electronics, St. Petersburg State Polytechnical University , St. Petersburg , Russia.
⁴ Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Science , St. Petersburg , Russia ; Department of Biophysics, St. Petersburg State Polytechnical University , St. Petersburg , Russia.
⁵ Laboratory of Nanobiotechnologies, Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine , Kiev , Ukraine.

Abstract

The mutant form GGBP/H152C of the D-glucose/D-galactose-binding protein with the solvatochromic dye BADAN linked to cysteine residue Cys 152 can be used as a potential base for a sensitive element of glucose biosensor system. We investigated the influence of various external factors on the physical-chemical properties of GGBP/H152C-BADAN and its complex with glucose. The high affinity (Kd = 8.5 µM) and high binding rate of glucose make GGBP/H152C-BADAN a good candidate to determine the sugar content in biological fluids extracted using transdermal techniques. It was shown that changes in the ionic strength and pH of solution within the physiological range did not have a significant influence on the fluorescent characteristics of GGBP/H152C-BADAN. The mutant form GGBP/H152C has relatively low resistance to denaturation action of GdnHCl and urea. This result emphasizes the need to find more stable proteins for the creation of a sensitive element for a glucose biosensor system.

Keywords: Biosensors; D-glucose/D-galactose-binding protein; Fluorescent dye BADAN; Glucose binding constant; Protein stability.

Grants and funding

This work was supported in part by the Ministry of Education and Science of the Russian Federation (Agreements number 8830 and 14.132.21.311), the Scholarships from the President of RF (SP-2390.2012.4, SP-563.2012.4), and the Russian Foundation for Basic Research (grant 12-04-31708 mol_a), Program MCB RAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.