Reaction of trypsin with 

 organophosphorus esters 



T. VISWANATHA 



Carlsberg Laboratory, Copenhagen, Denmark 



The relationship between protein structure and biological activity has been 

 a matter of considerable discussion in recent years. Turning our attention 

 to specific cases of proteolytic enzymes, the opinion as to the nature of the 

 active site(s) appears to be sharply divided. Thus, for example, in the case 

 of a-chymotrypsin, photo-oxidation studies by Weil and co-workers,^ the 

 dye-binding studies of Hartley and his associates, ^ the reaction with fluoro- 

 dinitrobenzene by Whitaker and Jandorf,^ and similar studies by others,* 

 suggest the association of an imidazolyl residue on the enzyme with the 

 active site. Evidence to the contrary have been reported by Neurath and his 

 school^ and by Cohen and his associates.^ The ability of chymotrypsin to 

 react with diwopropyl fluorophosphonate (DFP) has been used in most of 

 the above-mentioned studies to throw some light on the nature of the active 

 site(s). 



Studies on the reaction of trypsin with DFP and diethyl j3-nitrophenyl 

 phosphate (E600) carried out in the Carlsberg Laboratory have led to the 

 following results and conclusions : 



(I) Both DFP and E600 can react with trypsin in the presence of high 

 concentrations of urea resulting in the incorporation of one mole of phos- 

 phorus per mole of the enzyme. A nearly stoicheiometric yield of/7-nitrophenol 

 (one mole per mole of enzyme) was obtained during the reaction with E600. 

 The unique feature of the reaction in the presence of urea is that the enzyme 

 maintains its ability to revert to the active form on appropriate dilution. 

 In aqueous medium irreversible inactivation of the enzyme accompanies the 

 reaction. 



(II) The reaction of trypsin with DFP in aqueous medium at pH 8-0 

 results in the incorporation of 2-0 moles of phosphorus per mole of the 

 enzyme. At pH more alkaline than 8-0, more than 2-0 moles of phosphorus 

 per mole of the enzyme are introduced into the protein. While the first 2-0 

 moles of phosphate introduced appear to be stable to acid treatment, the 

 additional phosphate groups are acid-labile suggesting their attachment to 

 amino groups on the protein. 



The presence of 2-0 moles of phosphorus has been investigated further. 



