Tritium labeling of proteins to high specific radioactivity by reduction methylation

The third component of human complement, hemoglobin A2, and IgG have been radiolabeled with tritium to specific activities of 179, 103, and 89 Ci/mmol, respectively, by reductive methylation. The labeling procedure is mild, requiring only brief exposure to formaldehyde (10 to 12 mM) and tritiated sodium borohydride (3 to 5 mM), and specific for the alpha amino groups of NH2-terminal residues and epsilon-amino groups of lysyl residues. The extent of modification for each protein ranged between 5 and 16% at available amino groups based on a stoichiometric reactivity of 2 mol of formaldehyde/mol of primary amine. Although the degree of substitution required to obtain these specific activities was high, no significant loss of antigenic or biological activities was apparent. Each labeled protein was 90 to 95% precipitable with antiserum elicited to the unreacted protein. The hemolytic activity of C3 was reduced by only 16%; the oxygen affinity of HbA2 was minimally decreased, and the phosphorylcholine binding affinity of an IgA myeloma protein (TEPC-15) was not significantly affected. Precise double antibody radioimmunoassays were developed with each tritiated protein capable of detecting less than 10(-8) g of antigen. Labeled protein samples have been stored for time periods up to 1 year without appreciable deterioration.