title: The effect of pH on the glucose response of the glucose-galactose binding protein L255C labeled with Acrylodan.
abstract: The glucose-galactose binding protein (GGBP) is used as an optical biosensor in medical and bioprocess applications. This paper investigates the effect of pH on the behavior of GGBP-L255C labeled with Acrylodan for the purpose of finding the optimum conditions for sensing purposes as well as for protein preparation, purification and storage. The Acrylodan-GGBP fluorescence response in absence and presence of glucose was measured under varying buffer and pH conditions. Dissociation constants (Kd) and Gibbs free energies (ΔG) for the protein-glucose binding were calculated. Binding was found to be energetically favored at slightly acidic to neutral conditions, specifically close to the pI of GBP (∼ 5.0). Minimal fluorescence response to glucose was exhibited at pH 3.0 accompanied by a blue shift in the steady state fluorescence spectrum. In contrast, an almost 45% response to glucose was shown at pH 4.5-9.0 with a 13-nm red shift. Frequency domain lifetime measurements and quenching with KI suggest that at highly acidic conditions both the glucose-free and the glucose-bound protein are in a conformation distinct from those observed at higher pH values.
authors.affiliation: Center for Advanced Sensor Technology, Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, United States. Electronic address: mayyada@aucegypt.edu.
authors.forename: Mayyada M H
authors.initials: MM
authors.lastname: El-Sayed
authors.affiliation: Center for Advanced Sensor Technology, Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, United States. Electronic address: sbrown12@umbc.edu.
authors.forename: Sheniqua R
authors.initials: SR
authors.lastname: Brown
authors.affiliation: Center for Advanced Sensor Technology, Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, United States. Electronic address: karunam@umbc.edu.
authors.forename: KarunaSri
authors.initials: K
authors.lastname: Mupparapu
authors.affiliation: Center for Advanced Sensor Technology, Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, United States. Electronic address: leah@umbc.edu.
authors.forename: Leah
authors.initials: L
authors.lastname: Tolosa
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; C038518:acrylodan; D015081:2-Naphthylamine; D005947:Glucose
keywords: Biosensor; Dissociation constant; Fluorescence; Glucose-binding protein; Lifetime measurements; pH effect
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