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 (οG) 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.