Proteins spectroscopic estimation

The coupling reaction releases free imidazole which absorbs UV light at 280 nm, interfering with spectroscopic estimation of protein coupling. 

Different techniques have been applied to study the protein-protein, protein-ligand and, in particular, MIP-ligand interactions. They may serve to estimate or determine the binding constant and the number of independent binding sites (N) of a ligand-to-receptor (MIP or antibody) interaction. The range of affinity constants that can be calculated depends on the sensitivity of the assay and, in those cases where the separation of the bound and free species is a step of the assay, perturbation of the equilibria in the separation step will also be important [22]. Direct nonseparation techniques such as spectroscopic techniques (e.g., SPR or fluorescence polarization) can be used as well as indirect separation techniques such as radiolabeling [22]. 

The advent of recombinant DNA technology has led to an increased interest in the structural characterization of proteins by spectroscopic methods. Few spectroscopic techniques can provide the amount of information regarding protein secondary and tertiary structure which can be obtained from circular dichroism (CD) spectroscopy. In this chapter we describe the capabilities of CD spectroscopy to provide details on the globular structure of proteins. In addition, we will provide an overview of quantitative secondary structure estimates via CD spectroscopy and of specialized CD methods for studying proteins in contact with membranes and other biomolecules. Certain aspects of protein CD spectroscopy have been previously reviewed [1-19]. 

The apparent molecular mass of D-Ser toxin was dramatically increased by the addition of guanidine hydrochloride to the elution buffer, although that of the L-Ser toxin was not altered by the denaturing reagent. In the presence of 5.2 M guanidine hydrochloride, the D-form toxin was eluted at the same position as the L-form toxin and the apparent molecular masses of the two toxins were estimated as 6 kDa based on calibration with the standard proteins. CD and fluorescence spectroscopic analyses revealed that the two toxins were unfolded and lost their secondary and tertiary structure in 5.2 M guanidine hydrochloride at pH 7.4, as described below. It, therefore, appears that the D-Ser toxin forms a compact folded structure, whereas the L-Ser toxin has a relatively unfolded or extended structure. 

Cd(II) plastocyanin does not, under the given experimental conditions. This is an illustration of an application of PAC spectroscopy to the study of protein-protein interactions, and it is even possible to estimate the dissociation constant directly from the spectroscopic data. Note that in this example the perturbation function and not the Fourier transformed data, is displayed in the figure, as the perturbation function illustrates the effect of dynamics more clearly. Note that the data analysis is almost always carried out on the perturbation function. 

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