Arylamines on Phenolic Compounds

Dissolve the protein-containing tyrosine residues (or another phenolic macromolecule) in 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4, at a concentration of 2-4 mg/ml. 

With stirring, add to each milliliter of the protein solution, 20 pul of 0.15 M tetranitromethane in 95 % ethanol (Sigma). Make the addition in small aliquots if more than several milliliters of solution are to be derivatized. 

Quench the reaction by immediate gel filtration using a column of Sephadex G-25 (Pharmacia). Equilibrate the column and perform the chromatography using 0.2 A1 sodium borate, pH 9, so that the protein will be at the proper pH for the reduction step. After the separation, a determination of the modification level may be done by measuring its absorbance at 428 nm. 

Add sufScient sodium dithionite to bring the final concentration in the reaction medium to 0.1 M. 

The formation of a diazonium group from the arylamine derivative can be done by treatment with sodium nitrate in HCl (see protocol in Chapter 9, Section 6.1). 

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