A method has been developed for labeling proteins by acetylation without appreciable loss in biological activity.
The commercially available tritium-labeled enzymes, prepared by exposure to tritium gas, have reduced enzymic activity (Worthington). We tried to label enzyme and other protein molecules under controlled conditions without affecting their biological activity. This was done through acetylation with labeled acetic anhydride. On the average, between one and five acetyl groups could be introduced per molecule of trypsin or RNase without affecting the enzymic activity. No damage was done to the biological activity of chymotrypsin and concanavaline when up to two acetyl groups per molecule were introduced. An efficiency of acetylation up to 50% can be achieved, depending on the degree of acetylation and on the protein, and in particular, on the experimental conditions.
The proteins were dialyzed against an aqueous solution at an ionic strength and pH a t which the protein is most stable in its native form. The dialyzed material was freezedried. The freeze-dried proteins were acetylated while in the solid state. The acetic anhydride has to be applied a t a very slow rate to the protein in order to maintain a uniform acetylation through the solid material. This can be done by maintaining the freeze-dried enzymes with benzene or an other solvent containing the labeled acetic anhydride. After complete wetting, the material was stored for several days a t a temperature at which the solvent (&4"C for benzene) crystallizes. This increases the