I have done my uv-visible experiment where native HSA give its peak at 280 nm..bt after adding drug the peak maximum is at 250 nm instead of 280 nm..what is the reason behind this ?
One possibility is that the drug has an absorbance peak at about 250 nm. You can easily check this by taking an absorbance spectrum of the drug alone. If that is the problem, you can subtract the drug spectrum from the drug+HSA spectrum to get the HSA spectrum for HSA with the drug bound. Then subtract from that the spectrum of HSA alone to get the difference spectrum. (However, UV absorbance spectroscopy isn't a very sensitive way of measuring drug binding to proteins. Consider using tryptophan fluorescence spectroscopy.)
A second possibility is that the drug is insoluble at the concentration added, making the sample turbid. This will appear as an absorbance that becomes greater as the wavelength decreases.
A third possibility is that the drug acts as a denaturant of the enzyme, causing it to aggregate. Aggregated protein has increased turbidity (see above).
thank u Adam B Shapiro
for ur ans...i will do fluorescence.
...the solution may be not turbid as its solubility in water is 200 mg/ml..
is there any method to understand whether the sol is turbid or not? .
The best way is to use an instrument called a nephelometer that measures the light that has been scattered by the sample. Another way is to chose the shortest wavelength at which the substance has an extinction coefficient of zero, then take a reading and see if you get an absorbance higher than the blank.
+Adam B Shapiro thank u for ur prompt answer
can we also check the turbidity by DLS (dynamic light scattering ) method ?
Yes, DLS can be used to detect turbidity. In fact, it is so sensitive to turbidity that the detector can easily be saturated by a slight turbidity. Assuming you get a usable signal, just use the signal intensity as your measurement, not the particle size distribution.
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