The enzymic hydrolysis of organophosphorus compounds

SIS OF O R G A N O P H O S P H O R by S- B E R T I L A U G U S T I N S S O N

vy and Biochemistry, University o/Stock

n of cholinesterase inhibition b y organc ompounds )f the m o s t active of these enzyme int lzymically dies, e n z y m e s exist t h a t catalyse the h,. isopropylt h y l - p - n i t r o p h e n y l p h o s p h a t e 4. These have been med in o u r l a b o r a t o r y and extended to several organophospl ially those )horus com I ining a P - N b o n d ; in the first place the d i m e t h y l a m i d o - e t h o x y - p h o s p or t a b u n iterature, see ref. 5) has been used as s u b s t r a t e in these enz:y m e studi¢ obtained "icily reported. l'here exists in the p l a s m a of various animal species (rabbit, (rabbi" dog, hol r a t , COW, uinea pig) an enzyme, t e n t a t i v e l y called " p h o s p h a t a s e " , which wt hydro ['he r a b b i t La is p a r t i c u l a r l y active. Using h u m a n blood serum, the "'pphhooss p h a t a s e " ] rated from testerase a n d p a r t l y purified. Among tissues (rabbit, pig, cow), kidney, adrenal gland, am and liver cau e enzymic @sis of t a b u n . Especially active is the adrenal gland of rabbit, rab the c( ore active Lhe medulla. " P h o s p h a t a s e " activity has also been observed for f, lung, br Lintestine. .rations k n o w n to contain acetylcholinesterase as the only eesterase (e. I sue, cobra n) do n o t hydrotyse t a b u n or o t h e r o r g a n o p h o s p h o r u s c o m p)ounds. Hu "ytes have " p h o s p h a t a s e " activity. Fhe '.'phosphatase" of h u m a n s e r u m is n o t identical with the tt: D F P hy y m e from y or adrenal gland. There is, for instance, no activation b y Mn ++ o: m z y m e as 1red w i t h the " p h o s p h a t a s e " of kidney (cf. ref.3). ~ystems containing cholinesterase a n d " p h o s p h a t a s e " as eI1zynle eenn z y m e illlXEUrej mix aDO aGeEylCllOll tylcholine m o r g a n o p h o s p h o r u s c o m p o u n d as s u b s t r a t e - i n h i b i t o r mixtLure have been studied in deta dl. s found, a m o n g o t h e r things, t h a t the presence of "phosph~ ~hatase", e.g., in h u m a n serum, has h a definite influence on the inhibition (inactivation) of cholinesterase cholinest b y o r g a n o p h o s p h o r u s co~m p o u n dIs. s . T h u s tile pls0 value of t a b u n for the inhibition of h huma~ u m a n s e r u m cholinesterase is 7.89 for t the oriiginal serum, a n d 8.31 w h e n "'phosphatase" is absent. Physos (I0 3 M y s o s t i g m i n e and prostigmine (io ; ) do n o t affect the " p h o s p h a t a s e " activity. Durin During the hydrolysis of t a b u n b y the action of " p h o s p)batase", batas h y d r o c y a n i c acid is liberated. liberate l t is k n o w rn t h a t this reaction takes place s p o n t a n e o u s l y• in alkaline solution 6. The a m o u n t of cyani 7anide parallels els the a m o u n t of carbon dioxide evolved from a bicarbonat ,rbonate s y s t e m b o t h for enzymic and a s p o n t a~neous n e o u s hydrolysis. The CO 2 liberation is due to the strongly acid reaction product, dimeth,ylamido-ethox~ , - e t h o x y - h y d r o x y - p h o s p h i n e oxide. One mole of this acid (de determined b y the COz productic :oduction) is formed ned per mole t a b u n hydrolysed. There is no release of dim,ethylamine, ethanol, or inorgar ~nic phosphate. Some general properties of the " p h o s p h a t a s e " have been determined, such as its bbehavio ehaviour in dialysis, p H stability ( o p t i m uam m stability at p H 6.5-7.5) , and the MICHAELIS c o n s t a n t for the t hydrolysis of t a b u n b y purified (ppK Kss = 2.67) and crude e n z y m e ( p K s = 2.46) of h u m a n serum. Specificity studies have s h o wn w n t h a t tile chloride analogue of t a b u n is hydrolysed nmre rapid pidly t h a n the cyanide analogue, also t h a t the m e t h o x y c o m p o u n d is hydrolysed at the highest ra rate c o m p a r e d with its higher h o m o l m ogues. O t h e r more generally k n o w n o r g a n o p h o s p h o r u s compoune :)ounds, such as D F P , T E P P , mintacol, etc. tc., h a v e been included in these studies. Acetylcholinesterase completel ~tely or almost completely inhibited b y t a b u n was reactivated 1b y incubation with " p h o s p h a t a s e " . This reaction is supposed to be similar to the reaction m ention nentioned above in which cyanide is split off )ff from t a b u n . The p h o s p h o r y l a t i o n of cholinesterase, the reacti~ .ction which is responsible for the inactivation tivation of t h a t enzymeV, m o s t certainly takes place at a hydro: ydroxy g r o u p (of serine, or one of the phenolic g r o u p s of a d i h y d r o x y p h e n y l derivative; cf. ref.8). T The " p h o s p h a t a s e " c a n n o t split a P - N N bond, and therefore this t y p e of chemical b o n d is m o s t unlikely unlike to exist b e t w e e n the p h o s p h o r u s containing p a r t and the esteratic site of the inactivated choli .holinesterase complex. A full account of these investi stigations will be published later as a series of p a p e r s in .tcta C Che~ hem.

Hcand.

l am greatly indebted to M:iss GUNILLA HEI,~IBORGER for skillful technical assistance.

REFERENCES

) 271. • C H A N U T I N , ,1. ~io[. C/ggDl., 204 1. 13iol. Chem., 204 (1953) 837-

([953)

953) 117. U953) 33~9.53) 3 °6.

]. ,tin. Ckem. Soc., 75 ([953) 4628. aJzd., 6 (i()52) 959. Rece

4th, 1954

I D E N T I F I C A T I O N OF TWO M E;TABOLITIES T A B O L I T I E OF I.~ N I C O T I N O Y L G L Y C I N E AND I - I S O N I C O T I N O)YL-e-ACE Y

'~AZINE)

BY P A P E R C H R O M A T O G R A P H Y I N RAT UF 1)V

A. D E F R A N C E S C H [ AND V. Z A M B O N I

Carlo 17.rba Tkerapeutic Researck i*~stitttte, Laboratory o/:3]icrobioIog~, M

)

iazid) and tinic acid hy During the c o m p a r a t i v e s t u d y of the metabolism of isonicotil n injected otinoyl-hydrazino-methanesulfonie acid (Neotizide), the urine urim of rats, v compound :hese t w o drugs, was examined b y p a p e r c h r o m a t o g r a p h y . I t was found pound X) w a s excreted, besides isoniazid and isouicotinic acid aci 1. described, Fhis s u b s t a n c e was detected on t h e c h r o m a t o g r a n l n o t onlyt bt y the m e t h o d s already describe Mineralight Lamp), and yellow colour after aft s, b y a b s o r p t i o n of s h o r t w a v e - l e n g t h U.V. light (MineraligL t h a t is, CtlOn stror blue violet colour after reacti t r e a t maent e n t with cyanogen bromide and a m m o n i a 1, b u t also b y strong colour it gives when treated treat benzidine and cyanogen bromide 2, and b y the characteristic green i w i t h be w i t h picryl chloride a n d a m m o n i a a. because isonicotinamide as we 'supposed at first, beca~ I~t was seen at once t h a t C o m p o u n d X was n o t isonicotinamide, values with the solvents tried. t h e c hl r o m a t o g r a p h i c b a n d of the latter has cleariy different R F vz ac IItt was t h e n t h o u g h t b y analogy w i t h t h a t which occurs in the m e t a b o l i s m of nicotinic acid acid), t h a t c o m p o u n d X n (whicha is p a r t l y eliminated in the urine combined w i t h glycine as nicotinuric 1 y f u r t h e r research. I n fact, w h e n m i g h t be isonicotinoylglycine (I). This hypothesis w a s confirmed b c o m p a~ring r m g the R F values observed by p a p e r c h r o m a t o g r a p h y in different solvents, c o m p o u n d X aHRb y the m e t h o d of Ron )und to b e h a v e as isonicotinoytglycine. This substance was prepared ic was found )eared ~ _ nicotinoylglycine. -;~; ..... 1-1.,~;n. A lon ~ l l a ocolours C d ~ l l ~ resulting r O ¢ l l l l - l n c * {from r K i m ttrroenaf m a l e n t with different reagents appear the tm LZCH4 gfor Also en colour w i t h picryl chloride and amnlonia. to correspond, especially the green F u r t h e r confirmation was g,dven when, after elution of the chronlatographic b a n d detected l)y aate was hydrolysed with 6 N hydrochloric acid for four hours in a b s o r p t i o n of U.V. light, the eluate sealed t u b e at 12o ° . Two p a p e r• c h r o m a t o g r a m s of the hydrolysate were run : one using as a solw' e n t n-butanol, s a t u r a t e d w i t h o. 5 N a q u e o u s acetic acid, and using as detecting reagents benzidine aand detecting cyanogen bromide; and a n o t h e r using phenol: w a t e r (8o:2o) as a solvent, and n i n h y d r i n as detecti rclne. ~ram isonieotinic acid was found ; in the latter c h r o m a t o g r a m glycil reagent. I n the former chromatog~ C O - - N H - - C H 2--COOH I

{

\N /

I

(I)

CO--NH--NH--COCH a /)-.

(N)

(H)

search. of isoniazid was detected in tim course of this resear, A n o t h e r p r o d u c t of the m eetabolism tabc used This is a s u b s t a n c e which was ntot o t noticed in the early e x p e r i m e n t s 1 because w i t h the solvent us i (isoamyl alcohol s a t u r a t e d with o. 5 N acetic acid) its c h r o m a t o g r a p h i c b a n d overlaps t h a t of 1SO)ound nicotinic acid. On the o t h e r h a n dd, using a m i x t u r e of isopropanol and w a t e r (85:I5) the compou micotinoyl ther metabolites of the isoniazid, and was identified as T-isonicotin w a s clearly separated from the othe