For spectrophotometric determination of inhibition of protein(BSA and Albumin) denaturation, what is the reason behind taking absorbance at 416nm and 660nm ? I understand that at 660nm we measure the turbidity but why do we measure absorbance at near UV spectrum(416nm) ? Few article suggests 416nm as absorbance spectrum whereas few other articles suggest 660nm.
As far as I am concerned，almost every biomacromolecule has strong UV absorbance，in which UV light cannot propagate more than 2~3 mm in biological tissue. By the way human eye is most sensitive to light with wavelength around 400nm.
I don't understand what you mean by this:
For spectrophotometric determination of protein(BSA and Albumin) inhibition
BSA is bovine serum albumin. It does not have catalytic activity. What sort of inhibition are you measuring? What assay are you performing?
Dear Abhik Mojumdar
In this experiment, turbidity was measured at 660 nm. You can also select the wavelength using the scan spectrophotometer. Run your sample detect the best wavelength and analysis percentage of inhibition of albumin denaturation and find the IC 50 value of your sample.
I'm really sorry Adam B Shapiro sir. I made a mistake in framing the question. I meant inhibition of protein denaturation.
Turbidity increases very steeply as the wavelength decreases. The protein itself has no absorbance at either wavelength, so both wavelengths measure turbidity. The shorter wavelength measurement is more sensitive at detecting turbidity. However, depending on the protein concentration, you might choose the less sensitive measurement to avoid exceeding the dynamic range of the instrument.
Thank you Adam B Shapiro, Manoj Wijesekara and Ziyang Wang for your valuable answers.
Regards
In addition to absorbance, photon scattering also plays a pivotal role in light propagating, which decreases with increasing wavelength. In other words, the turbidity should be decreased by diluting the protein solution to a degree where lambert-beer law works well, and then you will get the real absorbance data.
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