Da Yu Protein Sciences – Biophysical Characterization
UV Derivative Spectroscopy - The microenvironment surrounding the amino acids tryptophan, tyrosine and phenylalanine is influential on their absorbance of ultraviolet (UV) light. Aromatic amino acids located within the protein’s hydrophobic core are in a less polar milieu than those exposed on the protein’s surface. The difference in the polarity of these two microenvironments has a significant effect on the UV absorbance of the aromatic amino acids. These polarity differences at times can lead to spectral shifts of as much as 2-4 nm. These shifts are best measured using either the second or fourth derivative of the zero order UV spectra. Shown on the left are the fourth derivative spectra for phenylalanine and tyrosine. What stands out in these spectra is the wealth of additional information they provide over the zero order spectra. Each derivative spectrum has a number of minima and maxima bands. It is the wavelengths of these bands that reflect the microenvironment of the aromatic amino acid and shifts in these wavelengths indicate changes in a protein’s tertiary structure.
In UV derivative spectroscopy each protein has its own unique spectral signature that is dependent on both its content of aromatic amino acids and the microenvironment surrounding those amino acids. In the fourth derivative spectrum shown on the right, the tryptophan residues in this protein's spectral signature provide the strongest signal, but distinct signals from phenyalanine and mixed signals from tyrosine are also observed.
Subtle changes in a protein's conformation are readily detected by UV derivative spectroscopy. As an example, protein unfolding by chemical denaturantion (e.g., urea, guanidine-HCl) significantly alters the fourth derivative UV spectrum of a protein. In the spectra shown above, as many as 11 maxima peaks are altered due to chemically-induced protein unfolding. Some maxima peaks shift by >1.5 nm indicating significant changes in the protein's tertiary structure.
With a resolution approaching 0.01 nm and the existence of multiple maxima, minima and inflections points in a protein's spectral signature, UV derivative spectroscopy is both a sensitive and powerful technique for monitoring the conformational integrity of therapeutic proteins.
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