Ribonuclease tritium labeling

Figure 3. Effect of ammonia on tritium distributions of (3-la-beled ribonuclease and lysozyme and on y-labeled ribonuclease.

The extremely large fraction of the total tritium in phenylalanine and tyrosine for /3-labeling of ribonuclease and chymotrypsinogen with TI can be understood in terms of a mechanism involving the addition of iodine atoms to aromatic rings, followed by reaction of the carbon radical with TI to give the cyclohexadiene derivative, which will tend to aromatize with loss of HI or TI to yield the tritiated parent amino acid as shown below. 

The same tritium distribution is obtained when ribonuclease is dried for a week in an evacuated drying pistol over P2O5 at 100 °C. before /3-labeling with TI. This experiment indicates that the mechanism does not involve exchange with TI dissolved in small amounts of protein-bound water. 

Figure 4. (y + ft)-, /3-, and y-labeled tritium distributions in ribonuclease with TI... 

Figure 6. y-Labeled tritium distribution of ribonuclease and chymotrypsinogen with HST and TI as radical interceptors... 

Tritium Distribution of Native Ribonuclease. In a standard y-label-ing experiment 20 mg. of protein were irradiated at 10 3 torr to about 5 Mrads at room temperature and subsequently exposed to HST at a pressure of about 150 torr for 4 hours (Experiment A, Figure 8). A portion of the unfractionated protein was then hydrolyzed, after exchange, to determine the tritium distribution. The results in Figure 8 demonstrate that essentially the same tritium distribution is obtained under a variety of experimental conditions (B-F). In Experiment B, ribonuclease was irradiated at 77°K. and then allowed to warm to 298°K. before adding HST. The comparison of distributions from Experiments A and B (Figure 7) shows that the radicals formed by radiolysis at 298°K. are identical with the secondary radicals produced from the primary radicals, formed by radiolysis at 77 °K. and subsequent heating to 298 °K. The same conclusion was previously reached (11, 22) from interpretation of ESR spectra. 

Normalized Specific Activities Among Native Proteins. Figure 9 shows a comparison of tritium distributions for native proteins irradiated to about 6 Mrads (except for myoglobin which was irradiated to 23 Mrads). Each bar represents the average normalized specific activity of five separate labeling experiments for ribonuclease and of two for each of the other proteins. The tritium distributions have many similarities. The activities of proline and methionine are generally high. Lysine and histidine are heavily labeled in most proteins, while threonine and serine... 

Each protein has, within this general pattern, its own characteristic radical distribution. In ribonuclease, lysine exhibits a much higher activity than do the remaining amino acids. Lysine and methionine are the most heavily labeled residues in lysozyme. In myoglobin, histidine has the highest activity. Methionine is the most heavily labeled amino acid in chymotrypsinogen, as is proline in insulin. Despite the similarities, therefore, each native protein exhibits a characteristic tritium distribution. 

This limitation in depth of labeling under TA conditions has been exploited in the study of proteins and protein assemblages, where the method is sometimes called tritium planigraphy. Such macromolecular systems, when treated under TA conditions are tritiated only in parts of molecules located at or very near the surface, leaving interior parts unlabeled. Subsequent dissection of the substrate, for example by separation of protein subunits or tryptic hydrolysis of protein followed by radioanalysis, can form the basis for inferences about the structure of the substrate. Applications of the TA method in this area include studies of the organization of the coat proteins of potato virus conformational dynamics of ribonuclease A spatial localization of coenzyme FAD in the quaternary structure of yeast alcohol dehydrogenase and subunit organization of the bacterial ribosome " . ... 

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