A method for measuring the rate of protein degradation is described. The method measures the change in 2-3H content of protein with time by racemization of the protein hydrolysate with acetic anhydride. The 3H on C-2 of amino acids is stable in proteins but becomes labile, owing to the action of transaminases, once the amino acids are released by proteolysis. The specific measurement of 2-3H in amino acids largely overcomes problems due to compartmentation and isotope recycling and evidence to support this claim is presented. Values for the half-life of Lemna minor (duckweed) protein determined by the new method are compared with values obtained by other methods.

A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the alpha-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the alpha-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.

Methods of measuring amino add recycling in Lemna minor are argued elsewhere (4) that the kinetic data of these experiments have been misinterpreted, because the kinetic treatment appropriate for a "label" experiment was applied to a "chase" experiment, so that the analysis not only gives the wrong rate, but the inverse of the true rate.Measurements of protein degradation based on tritium labeling of protein have shown that when Lemna minor is deprived of nitrogen its growth rate is greatly reduced and protein degradation is increased (10). This suggests an alternative strategy which the plant might employ to adapt to the deprivation of nitrate. The plant could change its enzyme complement by rapidly synthesizing new proteins from amino acids formed by degradation of old proteins.The reutilization of amino acids -amino acid recycling -is one of the main contributing factors to underestimating the rate of degradation of proteins. Protein half-lives are most frequently measured by labeling cells with radioactive amino acids and then following the decrease in trichloroacetic acid-precipitable radioactivity. The error in this method, which is produced by amino acid recycling, is well documented (13) and attempts have been made to evaluate the extent of amino acid recycling in animal tissues (3,16). Koch (14) concluded that "no matter how an isotope experiment is conducted the true half-life cannot be calculated without information additional to the time course of activity in the component under study."We have adapted the technique of density labeling pioneered by Varner (18)

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A method for measuring the rate of protein degradation in plant tissue is described. The method uses density labelling to avoid difficulties associated with compartmentation and recycling of amino acids. Although the technique cannot be readily adapted to measure the rate of degradation of single proteins, it avoids difficulties of interpretation due to enzyme activation or inactivation. Values for the half-life of Lemna minor protein obtained by this method are compared with values obtained by a number of other methods. To obtain satisfactory results it was necessary to improve the method of isopycnic centrifugation in CsCl gradients. A considerable improvement was achieved by using KBr gradients, and the advantages of using KBr rather than CsCl for the separation of density-labelled protein are discussed.