In this case, I am studying the interaction of a metal ion with a protein and want to know the effect of binding on the structure of the protein and conducting a concentration-dependent study. In the UV range both the species are absorbing and after subtracting the spectra, some part of the curve is going towards negative absorbance which is not valid.
I think in order to do a subtraction you need to know the binding constant. From the binding constant you can figure out amount of free protein present in the solution. If you subtract this value (absorbance) from the overall absorbance I guess you should be able to get absorption spectra of the metal ion bounded protein.
To know the binding constant you might take the help of UV-vis, NMR etc. This tutorial review might help you http://pubs.rsc.org/en/content/articlehtml/2011/CS/C0CS00062K
In Uv-vis you have to be careful concerning the nomenclature.
You have to concider the Lambert-Beer law and all of the mathematical laws which follow the use of this equation.
If you use absorption = 1 - transmission you can subtract the spectra (proportional to number of absorbed photons). For absorbance = log(1/T) I suggest to calculate the absorption.
Article About the possibility of calibrating optical detectors by so...
It would be better to do the experiment in a single setup than doing all those subtractions. I suppose you are using a split-beam UV visible spectrometer with a reference cell and sample cell. I would suggest you to add the titrant to both the cuvettes while titrating. If the titrant and analyte are non-interacting, you shall see no change in the absorption spectra (of protein). Similarly, if they are interacting, it would be reflected. No subtraction required. However, you have to be very careful about the dilutions in reference and sample cell (e.g. if you take 500ul buffer+50ul protein in the sample cell, you have to take 550ul of buffer in the reference cell).
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