Spectrophotometric assays for protein amino acid side chains

9 Spectrophotometric assays for protein amino acid side chains 

There are a number of useful reagents that allow the stoichiometry and reactivity of amino acid side chains to be monitored spectrophotometricaUy. Although these ate not always absolutely specific fiar the amino acid side chain chosen, the experimental protocol is straightforward and the inferences can normally be checked using other methodologies. Note that these reagents are hazardous and users should follow the appropriate laboratory safety regulations for their use. 

The classic reagent is 5,5 -dithiobis-(2-nitrobenzoic acid) or DTNB, sometimes known as Ellmans reagent (32), which is absolutely specific for the thiol side 

An alternative reagent for lysine residues is pyridoxal phosphate which reacts with improtonated amine groups to form a Schiff base. The reagent therefore modifies reactive lysyl amino groups at pH values around 7.5. The unstable imine can be reduced using sodium borohydride (added in aliquots to a final concentration of 1 to 10 mM after having adjusted the pH of the medium to 4.5 to 6) to form a stable adduct which absorbs at 325 nm (8325 = 9710 m cm ) and emits fluorescence at 425 nm. Further details can be foimd in (34). 

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