Labeling of proteins with [³⁵S]methionine and/or [³⁵S]cysteine in the absence of cells

Incubation of [³⁵S]methionine and [³⁵S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the ³⁵S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The ³⁵S labeling was resistant to dissociation by reducing SDS- PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [³⁵S]cysteine than [³⁵S]methionine. Incubation of ³⁵S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent ³⁵S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.