I mixed HSA with a compound. I found that after addition of the compound, the absorptivity of tryptophan (HSA) decreases. What can be reflected by this phenomenon?
You may have observed hypochromicity of Trp caused by its interaction with the compound. Have a look at this paper:
http://www.jbc.org/content/241/4/916.full.pdf
hope i did not get you wrong, if you mean that the abs(Trp) goes down - are you sure that the HSA concentation in solution is still the same after adding the compound? I would not be surprised if your protein precipitates if you add a compound.
Have you tried fluorescence measurements (trp-fluorescence at approx. 330nm, broad peak, don't get confused by the (small) raman peak that you will also see if you excite at e.g. 295nm)
But Adam is of course also right: changes in molar extinction coefficient due to local environment changes might be possible, too.
I would agree with Gregor that the precipitation is a likely cause of a decrease in the Trp absorbance. Another aspect is the accuracy of measurements. Does the compound absorb at 280 nm? What is the sample absorbance? Do you correct the absorbance at 280 for that of the added compound? What is the difference in the sample absorbance at 280 nm before and after adding the compound? Do you consider dilution of your sample as a possible cause for decreasing in the Trp absorbance?
Hi, Gregor and Alexander! Thanks for your nice suggestions! In my measurement, no precipitation could be observed. The compound has a little absorbance value at 280 nm. I didn't correct the absorbance at 280 nm. How can I do that? The dilution effect should be ignored because the total concentration of HSA in the measurement of individual HSA and the mixture of (HSA+compound) is same. The fluorescence measurement show a sufficient quenching of emission of Trp upon the addition of that compound.
Hi, Yin. To answer your question requires more information. Your experiment should be discussed in quantitative terms to make a conclusion on a cause for the observed effect. Terms like "more", "less", etc do not work here at all. Just read a good quality research paper (experimental section) to see what type of data is usually provided. In your case, it would be helpful to know experimental conditions, like concentrations of components, their spectra, instrument settings, how you conduct the experiment, etc. After this, it would be possible to conclude whether the observed effect is due to interaction between compounds or it is an artifact, error, or something else.
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