This appears to be an assay whereby proteolysis of BSA generates small TCA soluble fragments. When you add TCA, your absorbance will go down (relative to the starting absorbance of the BSA) because you are removing TCA-insoluble BSA and large protein fragments by centrifugation. It seems to me that you should be looking only at the increase in absorbance in the TCA supernatant. At t=0, (substantially) all the BSA should be precipitated with TCA and so your reference absorbance becomes zero (or close to zero). As TCA-soluble small fragments are generated by the protease, the absorbance for the supernatant should increase (though fragments without tyr will give little or no signal). The dramatic alteration in pH may also change the absorbance, as it will alter the ionization state of tyrosine.

There are far better ways of assaying proteases. I would consider peptides with reporter groups which become fluorescent upon cleavage by the protease. Depending on the exact requirements of the protease, this can involve removal of a coumarin-type dye from the peptide terminus which then fluoresces, or cleavage within a short peptide sequence to relieve the internal quenching of carefully matched reporters positioned at either side of the scissile bond.

Reading the 215-230 nm absorbance would say something about forming proteolytic peptides. You should follow de novo peptides rather than their precursor protein which is under a degradation pattern. Briefly, track the peptides by LC or spectrometric assays in total at 215-230 nm interval time by time, and do not stick yourself at 280nm within this experimental design.

@Nick Gee Thanks for the detailed explaination of the principle. As you said the absorbance should increase ideally, but that is not the case while I'm performing experiment.

The absorbance pattern is showing decreasing trend and therefore I'm unable to conclude anything from experiment. Could you please help me in knowing why its happening so?

@ismail Emir Akyildiz, Thankyou for your suggestion.

I followed your suggestion and took readings at 220nm but the absorbance is showing no increasing pattern.

Please provide some suggestions to improve the results.

It will be very helpful.

The assay as you describe it involves the production of TCA-soluble peptides. There are only two possible outcomes in an experiment. First, there is no proteolysis, in which case there will be no increase in signal compared to the control (i.e., without added protease). Second, there is some degradation of the BSA, in which case the concentration of TCA-soluble fragments will go up. There are no scenarios where the signal can go down.

I think you need to send your raw data, as it may be that your analysis of the data is incorrect, or the readings are too low or too variable to allow any valid conclusions to be drawn. It is also worth checking that your reader is not wrongly set to read transmittance rather than absorbance.