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6660908 
Journal Article 
Mode of inhibition of chymotrypsin by diisopropyl fluorophosphate. II. Introduction of isopropyl and elimination of fluorine as hydrogen fluoride 
Jansen, EF; Nutting, F; Jang, R; Balls, AK; , 
1950 
Yes 
Journal of Biological Chemistry
ISSN: 0021-9258
EISSN: 1083-351X 
185 
(1) 
209-220 
English 
15436492 
BCI:BCI19502400035260 
The isopropyl groups as well as the phosphate of diisopropyl fluorophos-phate (DFP) were found to have been introduced into the crystalline, inert protein resulting from the treatment of alpha-chymotrypsin with DFP. Two isopropyl groups were introduced per mole of enzyme. No F was found in the inert protein. Furthermore, acid (apparently HF) was formed during the inhibition reaction in the proportions of approx. 1 mole per mole of chymotrypsin. Therefore, DFP reacted with this enzyme by a condensation reaction with the liberation of the F as HF. The crystalline, inhibited protein had the same amino N content and electrophoretic mobility as alpha-chymotrypsin. There was thus no evidence that either an amino or a carboxyl group reacted with the DFP. None of the amino acids known to occur in chymotrypsinogen, even when used in relatively large amts., interfered with the inhibition of alpha-chymotrypsin by DFP. Furthermore, an acid hydrolysate of chymotrypsinogen (after neutralization) likewise did not interfere with the inhibition reaction. This did not mean that DFP did not react with an amino acid residue of chymotrypsin. Such a reaction was quite possible, but must then have depended on some special configuration in the protein, presumably one by virtue of which the protein was an enzyme. Contrary to preliminary results, DFP did not react with a "terminal threonine" of alpha-chymotrypsin. This terminal threonine was shown to be due to a small amt. of impurity in the alpha-chymotrypsin. The impurity was removed from the enzyme by treatment with DFP. It was isolated and examined. After crystallization inhibited chymotrypsin was free from this impurity. Relatively large amts. of tetraethyl pyrophos-phate inhibited alpha-chymotrypsin at a slow rate and trypsin at a still slower rate. || ABSTRACT AUTHORS: Authors