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Analysis of Photodegradation of proteins by UV-Vis Spectroscopy (CAT#: STEM-MB-0918-WXH)

Introduction

The exposure of proteins to light and the ensuing chemical and physical degradation has been studied extensively for many years. The residues in proteins that undergo primary photooxidation include tryptophan, tyrosine, phenylalanine, and cysteine/cystine. While photooxidation has been recognized as a major contributor to protein degradation, the effects of photoinduced damage have not been widely studied for biopharmaceuticals. This is particularly important since photodegradation can lead to changes in primary, secondary, and tertiary structures of protein and these changes, while not definitively established, could lead to differences in long-term stability, bioactivity, or immunogenicity.




Principle

UV-Vis spectroscopy is an analytical technique that measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample. This property is influenced by the sample composition, potentially providing information on what is in the sample and at what concentration. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.

Applications

UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of diverse analytes or sample, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.

Procedure

1. Calibrate the Spectrometer
2. Perform an Absorbance Spectrum
3. Kinetics Experiments with UV-Vis Spectroscopy

Materials

UV/VIS Spectrophotometer
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