Glucose-galactose binding protein (GGBP) is used as an optical biosensor in bioprocess and medical applications. This article investigates the effect of pH on the behaviour of Acrylodan-labeled GGBP-L255C in order to find optimal conditions for detection purposes, as well as for protein preparation, purification, and storage. The fluorescence response of Acrylodan-GGBP was measured in the absence and presence of glucose under different buffer and pH conditions. Dissociation constants (Kd) and Gibbs free energies (ΔG) were calculated for protein-glucose binding.
Binding was found to be energetically favoured under slightly acidic to neutral conditions, specifically near the pI of GBP (~ 5.0). A minimal fluorescence response to glucose was shown at pH 3.0 accompanied by a blue shift in the fluorescence spectrum at a steady state. In contrast, a nearly 45% response to glucose was shown at pH 4.5 to 9.0 with a redshift of 13 nm. Measurements of frequency domain shelf life and KI extinction suggest that under highly acidic conditions, both glucose-free and glucose-bound proteins have a different conformation from those seen at higher pH values.
glucose-binding protein, pH effect, fluorescence, dissociation constant, shelf life measurements, biosensor
Materials And Methods
D-Glucose, HEPES, Acetic acid, Tris HCl, Bis-Tris, Citric acid monohydrate (C6H8O7H2O), Boric acid (H3BO3), Dibasic sodium phosphate (HNa2O4P), Monobasic sodium phosphate monohydrate (H2NaO4PH2O) se purchased from Sigma Aldrich (St. Louis, MO).
- Preparation of biosensors and buffers
The fluorescently labelled GBP L255C biosensor was prepared as described in Ge et al. . Buffers were prepared based on the desired concentration and pH. To obtain the environment of the desired protein (L255C GBP), the protein was dialyzed in specific buffers. Specifically, 3 ml of purified GBP was placed in dialysis cassettes (Slide-A-Lyzer, Thermo Scientific) and the protein solution was dialyzed into 500 ml of citrate, acetate, HEPES, Bis-Tris, Tris, phosphate or borate for 14 hours (overnight) in a cold room.
The buffer was changed in the morning and the protein solution was dialyzed for a further 6 hours in a cold room. The ionic strength of all the buffers used was 20 mM. Protein solutions were collected, filtered with vacuum filters (Nalgene 0.2-micron vacuum filters, Fisher Scientific) to ensure the sterility of the buffer, and stored at room temperature in glass bottles for no more than 24 hours.
- Steady State GBP Fluorescence Measurements
Protein activity was determined using steady-state fluorescence spectra with a Varian Cary Eclipse fluorescence spectrophotometer. Emission spectra in the range 400 to 650 nm were recorded by keeping the excitation wavelength constant at 390 nm. A sample of GBP 250 µl (3.5 µM) was placed in a quartz cuvette and the fluorescence spectrum was measured.
The activity and emission spectrum were then checked by adding a 0.1 mM glucose standard to the cuvette to obtain a final concentration of 0.4 µM. After taking the reading, this standard was added two more times for final concentrations of 0.8 µM and 1.2 µM and fluorescence readings were taken for each. The glucose assay was completed by adding 1 mM, 10 mM and 1 M glucose standard, respectively, to the GBP sample in the same manner as described above. Final glucose concentrations after addition to the GBP solution ranged from 5.2 µM to 16 mM. The fluorescence reading was taken after each glucose addition.