The measurement of protein concentration by UV absorbance, such as at 280 nm, is based predominantly on the absorbance of tryptophan, tyrosine and phenylalanine side chains. Since these do not change when the protein is degraded, the concentration measurement is unaffected. This measurement should be made under strongly denaturing conditions such as 6 M guanidine HCl.

If you do not use guanidine, the protein may aggregate as it degrades because of the exposure of its hydrophobic core. This will result in turbidity (cloudiness), which is especially bad at UV wavelengths. The result will be a falsely high reading.

As the measurement depends on the chromophores of tryptophan, tyrosine, and phenylalanine they can be indeed influenced by denaturation. As the structure of the protein changes during denaturation also the chemical environment of these chromophores changes affecting their absorbance. However, I really doubt that you can use it for stability studies as the dynamic range should really small due to the increasing turbidity as Adam already mentioned and most photometers are not very accurate.

For stability assays, I recommend to use circular dichroism (CD) spectroscopy.

Dear Fatina

as Adam B Shapiro suggest, the UV vis determination depends from aminoacid composition which do not change when the protein is degraded.

Generally for mab you can try some different approach to investigate their stability:

- SDS page

- ELISA (check how the ability of the mab to bind it change during time or after stress)

- Size exclusion chromatography to detect aggregates and degrated fragments

SEC is the most expensive in terms of sample, while ELISA and SDS-page can be done with very limited amout.

good luck

Manuele