A simple method to determine fluorescence emission spectra of proteins in solid state is described. The available commercial accessories can only accommodate solid samples and hence do not allow a direct comparison between fluorescence spectra of a sample in solution and solid state form. Such comparisons are valuable to monitor the changes in protein structure when it is “dried” or immobilized on a solid surface (for biocatalysis or sensor applications). The commercially available accessories also do not allow working in a high throughput mode. We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples. This method works with a 96-well plate format. It enables the comparison of fluorescence spectra of a sample in a solid state with solution spectra, using comparable quantities of protein. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form and the lyophilized powder form. α-Chymotrypsin in solution showed a λmax of 335 nm while a high-activity preparation of the same enzyme for non-aqueous media, known as enzyme precipitated and rinsed with propanol (EPRP), showed an increase in the intensity of the fluorescence emission spectra. However, there was a small red shift of 2 nm (λmax of 337 nm) in contrast to lyophilized powder which showed a λmax of 328 nm. This is due to a difference in the tertiary structure of the protein as well as the microenvironment of aromatic residues between the two preparations. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. The relative fluorescence intensity of lyophilized GFP powder was decreased significantly to 17% as compared to GFP in solution, and showed a red shift of 4 nm in the emission λmax. It was found that fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP was significantly diminished. This indicated the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization.
Tuning the near infrared II emitting wavelength of small molecule dyes by single atom alteration
