THE APLICATION 

 OF DIRECT TRITIUM-LABELLING METHODS 

 TO THE CYTOCHEMISTRY OF PROTEINS 



E. A. BARNARD and J. MARBROOK 



Department of Zoology, University of London King's Collège, 

 Strand, London W.C.2. 



Isotopic labels can be employed with advantage in directiy-applied cyto- 

 chemical reagents forming covalent bonds at sites in proteins and other 

 macromolecules. The difficulties of introducing sufficient light-absorbing 

 structures, and the related problems of micro-spectrophotometry, can be 

 avoided hère. Thèse methods must be distinguished from those in which 

 the isotope is introduced metabolically prior to fixation, and also from 

 those in which a labelled enzymic reaction product is adsorbed. 



We hâve examined the application of small tritiated reagent molécules 

 to the quantitative cytochemistry of protein groups. High resolution, and 

 ample labelling after very short exposures, are obtained thus with tritium. 

 The factors involved in obtaining accurate quantitative results with such 

 reagents hâve been examined. While comparative measurements can bemade 

 readily, absolute measurements can also be obtained but only after cali- 

 bration of each system investigated. 



Measurements hâve been made on the total number of groups available 

 to acylation, and on the amino groups of proteins in varions situations. 

 Comparison with micro-spectrophotometric measurements using colour-form- 

 ing reagents can give valuable calibrations of the latter methods. 



The similar use of tritiated groups in labelling anti-bodies for cyto- 

 chemical application, e.g. for enzyme localisations, will also be discussed. 



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