Biochemical Techniques and Instrumentation

1.2.7. Applications

The usual procedure for colorimetric assays is to prepare a set of standards and produce a plot of concentration versus absorbance called calibration curve. This should be linear. Absorbances of unknown sample are then measured and their concentration interpolated from the linear region of the plot. The maximum absorbance should be approximately 0.5 to obtain good spectra.

Applications

1. Qualitative analyses are performed in the UV/Vis region to identify certain classes of compounds in pure state as well as in biological mixtures.
2. Quantification of biological samples either directly or via colorimetric assays is most common. Proteins are quantified directly using their intrinsic chromophores, tyrosine and tryptophan.
3. Difference spectroscopy detects small absorbance changes in systems with high background absorbance. A difference spectrum is obtained by subtracting one absorption spectrum from another. Common applications are determination of number of aromatic amino acids exposed to solvent, detection of conformational changes occurring in proteins, detection of aromatic amino acids in active sites of enzymes and monitoring of reactions involving catalytic chromophores.
4. Derivative spectroscopy differentiates the absolute absorption spectrum of a sample and differentially plot against the wavelength. The successful applications include the binding of a monoclonal antibody to its antigen with second order derivatives and quantification of tryptophan and tyrosine residues in proteins with fourth order derivatives.
5. Solvent perturbation is brought out by the change in polarity of solvent that causes a change in the UV spectrum of a protein without changing its conformation. It is used to probe the surface of a protein molecule.
6. Biochemical assays are performed based on the continuous monitoring of the absorbance of a system at a given wavelength throughout the course of the experiment. It is used for enzyme assays and kinetic analysis