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The influence of certain chemicals upon amylase activity Edwards, Howard I. 1934

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1  -  j ACC. P*0.  THE INFLUENCE OE CERTAIN CHEMICALS UPON AMYLASE ACTIVITY by Howard I . Edwards  *#««  A T h e s i s submitted f o r t h e Degree o f MASTER OP ARTS i n t h e Department o f CHEMISTRY  * * «•  The U n i v e r s i t y o f B r i t i s h Columbia April,  1934.  &£2?&Q^„^^  !  f  ACKNOWLEDGMENT , The w r i t e r wishes t o express h i s thanks t o Dr. R.H. C l a r k , Head o f t h e Department o f Chemistry, under whose d i r e c t i o n t h i s r e s e a r c h was c a r r i e d out, f o r t h e a d v i c e g i v e n and t h e i n t e r e s t shown d u r i n g t h e p r o g r e s s o f the v/ork;  t o Dr. A.H, H u t c h i n s o n ,  Head o f the Department o f Botany who so k i n d l y prov i d e d the greenhouse space n e c e s s a r y i n the germina t i o n experiments;  and t o Mr. H.S. McLeod, D i s t r i c t  I n s p e c t o r , Dominion Department o f A g r i c u l t u r e , who supplied  t h e C e r t i f i e d Seed P o t a t o e s used  experiments•  ###*«  i n these  TABLE OP CONTENTS INTRODUCTION P r e v i o u s work upon Dormancy in plants. Review o f t h e l i t e r a t u r e upon Amylase a c t i v i t y . D i s c u s s i o n o f methods o f measurement available. PART I The a m y l o c l a s t i c a c t i v i t y o f malt diastase. E x p e r i m e n t a l Methods Results Table I PART I I The e a c c h a r o g e n i c a c t i v i t y o f m a l t diastase. E x p e r i m e n t a l Methods Results Table I I Figure 1 . Figure 2 . PART I I I The e f f e c t o f c e r t a i n c h e m i c a l s upon dormant p o t a t o t u b e r s . E x p e r i m e n t a l Methods Results Table I I I DISCUSSION SUMMARY BIBLIOGRAPHY  1.  THE INFLUENCE OE CERTAIN CHEMICALS UPON AMYLASE ACTIVITY. ** **»*»*«• Introduction I n a p r e l i m i n a r y r e p o r t o f an i n v e s t i g a t i o n c a r r i e d out i n t h i s l a b o r a t o r y , C l a r k , F o w l e r and B l a c k potassium thiocyanate,  ethylene  (1) have shown t h a t  c h l o r o h y d r i n and t h i o u r e a  exert  a d e f i n i t e l y s t i m u l a t i n g e f f e c t upon t h e a c t i v i t y of m a l t d i a s ~ tase.  From t h e d a t a o b t a i n e d ,  i t was suggested t h a t t h e a b i l -  i t y o f t h e s e compounds t o induce a renewal o f growth i n dormant p l a n t s w a s due p r o b a b l y to t h e i r a c t i o n upon t h e enzymes concerA  ned  i n t h e u t i l i z a t i o n o f t h e food  growth and g e r m i n a t i o n ,  r e q u i r e d f o r the i n i t i a l  and more p a r t i c u l a r l y , t h e p l a n t amyl-  ases o In t h i s r e p o r t and i n a more recent paper by Denny ( 4 ) , i t i s s t a t e d however, t h a t no i n c r e a s e i n amylase a c t i v i t y was observed when such c h e m i c a l s were added d i r e c t l y to t h e f r e s h l y expressed p o t a t o j u i c e . e f f e c t of the thiocyanate  From t h i s , Denny c o n c l u d e s t h a t t h e and c h l o r o h y d r i n upon dormant p l a n t s  i s i n d i r e c t i n n a t u r e r a t h e r than a d i r e c t s t i m u l a t i o n o f the potato  amylase. In support o f t h i s c o n c l u s i o n , Denny f u r t h e r shows (5)  that ethylene  c h l o r o h y d r i n w h i l e h a v i n g no a p p r e c i a b l e  effect  when added t o t h e expressed j u i c e d i r e c t l y , d i d produce a defi n i t e increase  i n amylase a c t i v i t y when t h e j u i c e was expressed  from tubers t r e a t e d w i t h the chemical,  s e v e r a l days p r e v i o u s l y .  Potassium thiocyanate e x h i b i t e d a corresponding t h e case o f l e s s dormant p o t a t o e s .  effect only i n  He suggests t h a t t h e power  o f such compounds t o break dormancy may l i e i n t h e i r a b i l i t y to s t i m u l a t e t h e p l a n t c e l l s i n t o t h e f o r m a t i o n o f a g r e a t e r amount o f enzyme, r a t h e r t h a n t h e s t i m u l a t i o n o f t h a t a l r e a d y present. In a d d i t i o n t o t h e work c i t e d above, a c o n s i d e r a b l e number o f i n v e s t i g a t i o n s have been c a r r i e d out i n o t h e r phases o f amyl« ase a c t i v i t y .  As e a r l y as 1875» Nasse (6)  was an important  reported that there  and s p e c i f i c dependance i n t h e a c t i v i t y of f e r -  ments, upon t h e p r e s e n c e o f s a l t s , w h i l e P r e t i (7) amylases from d i f f e r e n t sources were rendered  reported that  practically  inert  by d i a l y s i s , but r e s t o r e d to a c t i v i t y by t h e a d d i t i o n o f neutral  electrolytes. A l a r g e p r o p o r t i o n o f t h e experiments have been c o n f i n e d  to t h e e f f e c t o f amino a c i d s and o t h e r p r o t e i n p r o d u c t s diastatic activity.  upon  Rockwood ( 8 ) has s t u d i e d t h e e f f e c t o f a  l a r g e number o f n i t r o g e n compounds on s a l i v a r y d i a s t a s e and noted t h a t t h e  amino a c i d s were a c c e l e r a t i n g i n c h a r a c t e r  w h i l e amides were n o t .  E f f r o n t (9) a l s o o b t a i n e d  u l t s and a s c r i b e d t h i s p r o p e r t y t o t h e i r amphoteric i n n e u t r a l i z i n g some i n h i b i t o r y product  character  of h y d r o l y s i s . Diastase,  g e n e r a l l y b e l i e v e d t o be p r o t e i n i n n a t u r e , es i n an aqueous s o l u t i o n .  s i m i l a r res-  rapidly deteriorat-  This l o s s i n a c t i v i t y i s considered  by Sherman and Walker (10) t o be due t o t h e h y d r o l y s i s o f the enzyme.  They suggest t h a t t h e f a v o r a b l e i n f l u e n c e exerted by  amino a c i d s i s due to the p r o t e c t i o n of the enzyme from h y d r o l ysis.  Sherman and N a y l o r (11) r e p o r t t h a t b e n z o i c a c i d , h i p p -  u r i c a c i d , a n i l i n e s u l f a t e and benzamide had l i t t l e t h e a c t i v i t y o f s a l i v a r y and p a n c r e a t i c amylase. the presence o f the c a r b o x y l and amino groups, er  i n t h e same m o l e c u l e , was  but o n l y compounds of t h e t h e r e was  Thus, n e i t h e r  alone, or togeth-  s u f f i c i e n t t o a c c e l e r a t e the enzyme,  amino t y p e were e f f e c t i v e .  some t r u t h i n RockwoocTs t h e o r y , was  Sherman a n d . C a l d w e l l (12).  e f f e c t upon  That  demonstrated  In t h e i r experiments they had  by found  t h a t amino a c i d s e x e r t e d a p r o t e c t i v e i n f l u e n c e a g a i n s t the i n h i b i t i o n o f p a n c r e a t i c amylase by m e r c u r i c c h l o r i d e . of  t h i s was o f f e r e d , but i f , as E u l e r suggests (13)  ITo explansuch t o x i c -  its'" i s caused by t h e c o m b i n a t i o n o f t h e m e r c u r i c i o n w i t h the enzyme m o l e c u l e , t h e amino a c i d s may  p r e v e n t t h i s by  combining  with the m e t a l l i c i o n . Up t o t h e p r e s e n t time however, l i t t l e  e x p e r i m e n t a l work  has been c a r r i e d out i n d e t e r m i n i n g t h e e f f e c t o f s p e c i f i c chemi c a l groups o r i o n s upon amylase a c t i v i t y . of  Moreover, a r e v i e w  t h e a v a i l a b l e l i t e r a t u r e has y i e l d e d c o m p a r a t i v e l y l i t t l e  inf-  o r m a t i o n c o n c e r n i n g the e f f e c t of the c o n c e n t r a t i o n o f the chemical  ' a c t i v a t o r s ' , upon t h e enzyme a c t i v i t y .  I t was  considered  t h e r e f o r e , t h a t such an i n v e s t i g a t i o n i n c l u d i n g a l a r g e v a r i e t y of  compounds over a wide range of c o n c e n t r a t i o n s , might prove o f  v a l u e i n t h r o w i n g f u r t h e r l i g h t on the mechanism o f enzyme r e a c t i o n s and t h e r e l a t i o n o f amylase a c t i v i t y t o dormancy. ition,  I n add-  because o f the d i v e r g e n c e i n r e s u l t s p r e s e n t e d i n (1)  i n a r e c e n t paper by Denny ( 1 4 ) , i t was  and  c o n s i d e r e d n e c e s s a r y to  repeat t h e experiments  w i t h p o t a s s i u m t h i o c y a n a t e and e t h y l e n e  c h l o r o h y d r i n and e x t e n d i n g them t o a w i d e r range o f c o n c e n t r a t ions. As a f u r t h e r t e s t of any r e l a t i o n e x i s t i n g between t h e abi l i t y of a compound t o break t h e r e s t p e r i o d o f dormant p l a n t s and  i t s e f f e c t upon d i a s t a t i c a c t i v i t y , t h o s e compounds  found  to be most a c c e l e r a t i n g i n c h a r a c t e r were employed i n an experiment to determine  t h e i r power i n b r e a k i n g the dormancy  o f pot-  ato t u b e r s . The u s u a l methods o f measuring  diastatic activity f a l l  into  two g e n e r a l c l a s s e s ; t h o s e d e t e r m i n i n g t h e r a t e o f d i s a p p e a r a n c e o f t h e s u b s t r a t e , and t h o s e d e t e r m i n i n g t h e q u a n t i t y o f r e a c t i o n p r o d u c t s formed.  Of t h e f i r s t t y p e , t h a t most commonly used i s  t h e Wohlgemuth method (15)» based upon t h e a l t e r a t i o n i n t h e c o l or reaction with iodine, occurring during starch hydrolysis. Measurements o f t h i s t y p e a r e an i n d e x o f t h e l i q u e f y i n g o r amylo c l a s t i c power o f t h e enzyme. p e r r e d u c t i o n procedure  Of t h e second  type, t h e u s u a l cop-  i s f r e q u e n t l y employed i n d e t e r m i n i n g  the r a t e o f formation of reducing substances.  T h i s i s an i n d e x  o f the s a c c h a r o g e n i c power o f t h e enzyme. Whether t h e r e s u l t s o b t a i n e d by t h e two methods a r e comparable i s s t i l l  a m a t t e r o f some c o n t r o v e r s y .  Most i n v e s t i g a t o r s  c l a i m t h a t d i a s t a t i c h y d r o l y s i s of s t a r c h i s e f f e c t e d by a t l e a s t two d i s t i n c t enzyme f r a c t i o n s o f d i a s t a s e . a l l attempts ful.  A6 y e t however,  to s e p a r a t e t h e two f r a c t i o n s have been unsuccess-  O h l s s o n (16) announced t h a t by a b r i e f h e a t i n g o f amylase  s o l u t i o n s , i t i s p o s s i b l e to destroy e n t i r e l y the s a c c h a r i f y i n g  component o f t h e system w h i l e y e t r e t a i n i n g the l i q u e f y i n g a c t ion.  I t i s p o s s i b l e t h a t i n t h e case o f amylase, instead, o f  two d i s t i n c t enzymes, t h e s i n g l e enzyme m o l e c u l e may c o n t a i n two s p e c i f i c r e a c t i v e groups, one concerned i n t h e d e g r a d a t i o n  of  s t a r c h t o d e x t r i n e and the o t h e r h y d r o l y s i n g t h e d e x t r i n s t o r e d ucing  bodies. Of t h e two methods of measurement, t h e d e t e r m i n a t i o n o f  the a m y l o c l a s t i c power may be t h e more b a s i c , s i n c e b e f o r e t h e formation of reducing  sugars can o c c u r ,  i t seems e v i d e n t  p a r t o f t h e s t a r c h a t l e a s t must f i r s t be h y d r o l y s e d ermediate d e x t r i n s .  that  to the i n t -  I n a d d i t i o n t o t h i s , t h e Wohlgemuth method  i s much more r a p i d l y e x e c u t e d .  S i n c e one o f t h e o b j e c t s o f t h i s  i n v e s t i g a t i o n was t h e t e s t i n g o f a l a r g e number of compounds, many of w h i c h might r e n d e r t h e more complex copper r e d u c t i o n i n a c c u r a t e o r even i m p o s s i b l e , er p r o c e d u r e , m o d i f i e d activity.  i t was d e c i d e d t o employ t h e form-  i n some d e t a i l s , as a measure o f amylase  However, f o r purposes o f comparison w i t h Denny's  r e s u l t s (14 ), t h o s e compounds found most a c c e l e r a t i n g were f u r :  t h e r t e s t e d u s i n g the s t a n d a r d  copper r e d u c t i o n p r o c e d u r e .  Part I The I n f l u e n c e of t h e C o n c e n t r a t i o n o f C e r t a i n Chemical Compounds upon t h e A m y l o c l a s t i c Acti v i t y of Malt Diastase. Experimental  Procedure:  As a s o u r c e o f t h e enzyme, malt d i a s t a s e , U.S.P. IX, prepared by Eimer and Amend, was used.  F o r - e a c h day's experiments,  1 .0 gram o f t h e enzyme m a t e r i a l was e x t r a c t e d f o r 30 minutes a t room temperature w i t h 5°° c c s . d i s t i l l e d water, a f t e r which a l l  6* s o l i d r e s i d u e was f i l t e r e d out as r a p i d l y as p o s s i b l e , a folded  through  filter.  As s u b s t r a t e , s o l u b l e s t a r c h ( B a k e r ' s , C .P.')• was employed, one l o t b e i n g used e x c l u s i v e l y throughout t h e e n t i r e i n v e s t i g a t ion.  As i n t h e case o f t h e enzyme e x t r a c t , a f r e s h  solution  was p r e p a r e d each day by m i x i n g 15»0 grams o f t h e s t a r c h w i t h 100  c c s d i s t i l l e d water and h e a t i n g i n a water b a t h t i l l  i o n was complete.  solut-  T h i s was f i n a l l y d i l u t e d t o a volume o f 200  ccs. The r e a c t i o n m i x t u r e c o n s i s t e d o f :  7»5 c c s . o f M c l l v a i n e '  c i t r i c a c i d - phosphate b u f f e r (17), 1»5 c c s . enzyme e x t r a c t , 6.66 c c s . s o l u b l e s t a r c h s o l u t i o n and v a r y i n g p r o p o r t i o n s o f a s o l u t i o n o f t h e c h e m i c a l under e x a m i n a t i o n . made up t o a volume o f 25*0 c c s .  The whole was then  This mixture a f t e r being p l a c  ed i n t e s t - t u b e s and corked, was i n c u b a t e d i n an e l e c t r i c oven a t a temperature o f 30°C»» f o r t h e c o u r s e o f t h e experiment. S t a r t i n g a p p r o x i m a t e l y two hours a f t e r t h e b e g i n n i n g o f t h e experiment, t h e r a t e o f d i s a p p e a r a n c e o f s t a r c h was observed by w i t h d r a w i n g a few drops o f t h e r e a c t i o n m i x t u r e from time t o time and t e s t i n g w i t h N/200 I o d i n e on a p o r c e l a i n spot p l a t e . The l e n g t h of t i m e r e q u i r e d f o r t h e h y d r o l y s i s t o r e a c h the achromic p o i n t , o r t h e p o i n t a t which no c o l o r o t h e r than t h a t o f t h e i o d i n e was apparent, was noted i n each case.  Sufficient  i o d i n e was added t o produce a w e l l - d e f i n e d c o l o r r e a c t i o n ,  alth-  ough c a r e was t a k e n i n i t s a d d i t i o n , f o r an excess obscured t h e end p o i n t • As Denny (14) and M i l l e r (18) have p o i n t e d out, c e r t a i n  compounds, p a r t i c u l a r l y p o t a s s i u m t h i o c y a n a t e w i t h t h e i o d i n e and inaccurate.  and  thiourea  react  are l i k e l y to r e n d e r the s t a r c h - i o d i n e t e s t  In t h e case of the t h i o c y a n a t e  i t was  found t h a t  by a d d i n g a few drops of d i l u t e h y d r o c h l o r i c a c i d to the m i x t u r e before  the i o d i n e , l i t t l e d i f f i c u l t y  was  experienced.  Thiourea  hov/ever, absorbed i o d i n e u n d e r a c i d and a l k a l i n e c o n d i t i o n s . I t was  found n e c e s s a r y to use a l a r g e r volume of t h e r e a c t i o n m i x t -  ure and  to add  s u f f i c i e n t N/50  i o d i n e s o l u t i o n to combine comp-  l e t e l y w i t h the t h i o u r e a b e f o r e the s t a r c h r e a c t i o n c o u l d be s e r v e d . Even under t h e s e c i r c u m s t a n c e s , o b t a i n accurate The  i t was  difficult  ob-  to  readings.  q u a n t i t y o f enzyme employed was  s u f f i c i e n t to c a r r y  t h e h y d r o l y s i s to the achromic p o i n t i n a p p r o x i m a t e l y f o u r h o u r s . I t was  considered  s i n g l e day, toluene,  more s a t i s f a c t o r y to complete each run i n a  i n o r d e r to a v o i d the use of p r e s e r v a t i v e s  which, a p a r t from i t s i n h i b i t o r y e f f e c t (19) might r e a c t  w i t h c e r t a i n o f the c h e m i c a l s i n use. a l s o i n d i c a t e d that constant was  such as  shaking,  P r e l i m i n a r y experiments p a r t i c u l a r l y when  toluene  added, decreased the r a t e of h y d r o l y s i s a p p r e c i a b l y , ( a p p o i n t  noted by S c h u l t z and L a n d i s (20)  i n t h e i r experiments w i t h veg-  etable amylases. Chemically  pure compounds were used t h r o u g h o u t r r a n d ^ a l l t  g l a s s w a r e a f t e r c l e a n i n g w i t h chromic a c i d , t h o r o u g h l y with d i s t i l l e d  water b e f o r e use.  A l l pH measurements were  made w i t h t h e quinhydrone e l e c t r o d e i n a l l but a few when the c o l o r i m e t r i c method was  rinsed  used.  instances  Compounds h a v i n g  too  g r e a t an e f f e c t upon the r e a c t i o n of the medium, even i n the  8. p r e s e n c e of the b u f f e r , were o m i t t e d from f u r t h e r t e s t s . Preliminary  experiments i n d i c a t e d t h a t f o r t h i s sample o f  m a l t d i a s t a s e a t l e a s t , the optimum r e a c t i o n range l a y between pH  6.0  pK 4.0  and pK 6.5, - 4.5  (21) .  a l t h o u g h the optimum range u s u a l l y g i v e n  is  I t i s q u i t e p r o b a b l p however, t h a t the opt-  imum r e a c t i o n v a r i e s w i t h the type of b u f f e r employed, as w e l l as w i t h the t e m p e r a t u r e . adjusted  toa  pH of 6.2.  (21) .  The  b u f f e r m i x t u r e used,  was  On a d d i t i o n of the s t a r c h s o l u t i o n to  t h e r e a c t i o n m i x t u r e , a PH of 6.4 v a r i a t i o n s were g r e a t e r t h a n  f i n a l l y resulted.  Ho  pH  ±.15• #**«*#  Results The  v a l u e s redorded i n Table I r e p r e s e n t the decrease ( a c c -  e l e r a t i o n ) or increase  ( i n h i b i t i o n ) i n time, i n minutes, r e q u i r -  ed f o r the h y d r o l y s i s of the s t a r c h i n the p r e s e n c e of the chemi c a l s l i s t e d , as compared to the c o n t r o l s .  The  r e q u i r e d f o r the appearance of the achromia p o i n t t u b e s was  a p p r o x i m a t e l y 245 m i n u t e s .  average t i m e i n the c o n t r o l  D a i l y v a r i a t i o n s i n the  a c t i v i t y of the enzyme were b a l a n c e d by comparing each day's experiments w i t h the c o n t r o l s f o r t h a t day o n l y . p r e s e n t e d a r e the mean of s e v e r a l r e a d i n g s . i n h i b i t i n g compounds had impossible  In cases where  so d e l a y e d the r e a c t i o n t h a t i t was  t o o b t a i n a v a l u e f o r the achromic p o i n t t h a t  the minimum degree o f i n h i b i t i o n i s r e c o r d e d . o n l y where h y d r o l y s i s was purple  A l l values  T h i s was  day, done  apparent, as evidenced by the red  c o l o r a t i o n o f the i n t e r m e d i a t e  In cases where the compound was  dextrins with  or  iodine.  so s t r o n g l y i n h i b i t i n g t h a t  no  d e x t r i n s c o u l d be d e t e c t e d by means o f t h e i o d i n e t e s t , b u t o n l y t h e b l u e c o l o r o f t h e s t a r c h was apparent, t h e e f f e c t i s r e c o r d ed as 'complete i n h i b i t i o n ' • TABLE  .1  V a r i a t i o n i n the a c t i v i t y of Malt Diastase i n t h e p r e s e n c e o f c e r t a i n c h e m i c a l compounds.  COMPOUND P o t a s s i u m Bromide  Potassium Chloride  CONC.  ACCEL. INK IB (Minutes)  0.01 0.03 0.10 1.0  100 121  %  0.0004  0.001  0.004  0.01 0.03 0.10 1.0 2.0  Potassium Iodide  0.01 0.03 0.10 •2.0  Potassium N i t r i t e  0.005 0.01 0.05 0.20  Sulfate  98  115 133 140  150 14  18 63 180  3.0  0.01  30  0.40  Potassium  0 35 80  35 55 87 102 120 137 125  2.0 Potassium N i t r a t e  120  0.03 0.10 0.60 0.01 0.03 0.10 0.60 1.0  41  34 15 0 0 2 13 12  10  COMPOUND Potassium  Thiocyanate  Ethylene Chiorohydrin  CQNC • _% 0.10  37  0.50 O.75 1.0 1.5 2.0  80 90 100 43 9  3300 0.24  17  0.60  1.20 2.48  3.72 4.96 6.20 6.80 7.44  Epichlorhydrin  ACCEL. INHIB. (Minutes).  38  38 55 71 80 80 53 7  78  0.80 1.6  2 16  2.4  Glycerol MoBichlorhydrin (Symmet.)  0.05 0.22  O.65  * .0 1 .64  Glycerol  ffionbehlbrhydrin  C h l o r a l Hydrate  Dichlorethylene  D i c h l o r e t h y l ether  Chloroform  0.26 1.32 4.0 5.30 8.0 0.20 1.0 2.0 0.025 0.10 .0375 0.23 0.46 1.20 0.06 0.27 0.90  42  31 72 96 120  1322  •  79 122  149  88 32 0 8 10 0 0 2 23 74 >130 6 0  0 39  11. COMPOUND  COHC . -HCSt  Ethyl  Iodide  E t h y l Bromide  n. P r o p y l  Bromide  Ethylene Glycol  Propylene Glycol  Trimethylene G l y c o l  Methyl  Alcohol  Ethyl  Alcohol  Allyl  Alcohol  AG GEL.  0.10  0.20 0.30 0.218 0.58 1 .16 1 .45 0.27 0.54 1 .36 0 .44 0 .90 2.23 0 .41 0.84 2.10 3.10 0.21 0 .42 1 .02 2.05 3.10 0.47 1 .60 6.38 15.90 0.80 3.16 9.50 15.8  3 11 9 12 16 22 6 22  0 2 2 12 1  10  14  22 29 5  3 1  6.90 17.10 1.58 3.16 6.32 9.50 15.80  34  160  1 .13  0.80  10  5 7  O.56  0.31  21  0  3.50  Acetone  INHIB  ^ B K l  0 20 30 23 2  6 13 120 8 15 52 > 120 > 120  12 135  12. COMPOUND Maltose  CONC.  0.20 0.50 1.00 2.00  3-GO Glucose  E t h y l Acetate  0  Brom E t h y l A c e t a t e  Alanine  0.50 1.0 2.0 3.0 0.90 1 .80 3-58 7.20 0.30 0.90 1 .50 3.0 0.08 0.24 0.40  Glyc ine  Tyrosine  Acetamide  1.20 0.10 0.50 1100  0.004  0.02 0.03 0.08 0.20  ACCEL.  15  14  11 5  0  2 5 58 53 47  10  19  2  10  20 9  13 16 16 27 46 1 6  0.40  0.08 0.20 0.60 1.20  21 >120  24 40  0.60 0.80 1 .20 0.08  0.04  5 10 34  91 118  0.80 1 .20 •Butyramide  30  40 77  0.40  Propionamide  1KB" IB  9  0  as 0 0 15 21 62  13COMPOUND Oxamide  Urea  Thiourea  CONC. 0.005 0.010  ACCEL 3 8  0.0375  26  0.10 0.50 1.00 1.50  11 3  0.10 0.50 1.00 2.00  9 20  20  48  77 63  P h e n y l Urea  0.015 0.04.5 0.225  0 6 25  Phenyl Thiourea  0.005 0.020 0.05 0.075  2 12 23 37  T o l y l Thiourea  0.01 0.10  12 38  M e t h y l Urea  0.16 0 .40 1 .20  26 39 56  Creatine Hydrate  0.04 0.20 0,60  10 24  Pyridine  0.20 0.80 5.90  0  12 Com]  Piperidine  0.070 0.17  Pyrrol  0.01 0.06 0.09 0.17  16 22 27 30  Succ i n i m i d e  0 .02 0.08  0  0.30  INHIB.  85 24 140  4 20  14.  COMPOUND Acetonitrile  SONC:W.  0.16 0.60  1.60 2.40  Phenol  o.  0.10  0.20 0.60 1.00  Chlorphenol  0 .012 0.062 0 .124  0.31 0 .62 0.93 0.10 0 .40 0.60 0.80  p. C r e s o l  Hydroquinone  Phioroglucinol  0.10 0.50 1.00 0 .04 0 .20 o.6§  ACCEL .  INK I B .  0 0 8  15 12 0  130  225 3  6  15 100 Complete I r i h i b . « « 40  80 >  120  >120  38 >1?0  Complete Inh i b . 17 100 >  200  Part I I  The i n f l u e n c e o f c e r t a i n c h e m i c a l s upon the s a c c h a r o g e n i c a c t i v i t y o f M a l t D i a s t a s e . Experimental  Procedure  The method employed d i f f e r e d f r o m t h a t d e s c r i b e d above o n l y i n d e c r e a s i n g the q u a n t i t y o f enzyme e x t r a c t added t o t h e  15r e a c t i o n m i x t u r e , from 1.5  toO.75 c c s .  The tubes c o n t a i n i n g t h e  h y d r o l y s i n g m i x t u r e were i n c u b a t e d at 30°C« f o r  a  period of four  h o u r s , r a t h e r t h a n t h e l o n g e r p e r i o d o f e i g h t e e n h o u r s used-by Denny (14) f o r t h e reason t h a t any a c c e l e r a t i o n due t o t h e added c h e m i c a l s would be more e v i d e n t i n t h e e a r l y s t a g e s of hydr o l y s i s t h a n when t h e r e a c t i o n had almost reached  equilibrium.  P r e l i m i n a r y experiments i n d i c a t e d t h a t t h i s was a c t u a l ^ t h e case. At the end of t h e f o u r hour p e r i o d , 5.0 c c . volumes of t h e r e a c t i o n m i x t u r e were p i p e t t e d i n t o 20.0  ccs. Fehling's  s o l u t i o n and t h e r e d u c i n g sugars d e t e r m i n e d by t h e s t a n d a r d Munson and Walker p r o c e d u r e (22).  Cuprous o x i d e was  by t h e v o l u m e t r i c permanganate method.  determined  Prom t h e cuprous o x i d e  v a l u e s thus o b t a i n e d were deducted t h o s e from b l a n k d e t e r m i n a t i o n s o b t a i n e d by the same p r o c e d u r e except t h a t 0.75  ccs. boiled  enzyme s o l u t i o n were added. I t was  found i m p o s s i b l e to a p p l y t h i s method to m i x t u r e s  c o n t a i n i n g t h i o u r e a however, owing t o the d e c o m p o s i t i o n of the l a t t e r compound on h e a t i n g w i t h F e h l i n g ' s s o l u t i o n w i t h the forma t i o n of a copper s u l f i d e .  Attempts to use t h e c o l o r i m e t r i c p i c -  r i c a c i d method were e q u a l l y u n s a t i s f a c t o r y . The v a l u e s o b t a i n e d a r e p r e s e n t e d below. Results In T a b l e I I Amylase a c t i v i t y i s expressed i n terms of m i l l i g r a m s o f cuprous o x i d e .  Each v a l u e g i v e n i s the mean of  s e v e r a l d e t e r m i n a t i o n s . As a f u r t h e r means o f comparison w i t h the Wohlgemuth method, t h e c o l o r s noted on the a d d i t i o n of i o d i n e  16. t o a few d r o p s o f t h e r e a c t i o n m i x t u r e a t t h e end o f t h e f o u r hour p e r i o d , a r e recorded. The e f f e c t o f t h e c o n c e n t r a t i o n o f c e r t a i n o f t h e compounds upon t h e a c t i v i t y o f t h e enzyme, i s r e p r e s e n t e d g r a p h i c a l l y i n F i g u r e s 1 and 2 » TABLE  COMPOUND  CONG. /o  I I  Mgs. Cu 0 2  Color Reaction  CONTROL  00  13.8  Blue  Alanine  0.10 0.20 0.^0 1 .00 1.50 1.80 3.6o  36.1 38.9 44.5 51.8 52.9 53.6 56 .4  Blue Blue v i o l e t Violet Violet Red v i o l e t V i o l e t red Red  0.015 0.06 0.15 0.30 0.45 0.60 1.20 1.50  38.5 40.4 42.2 46.5 50.2 49.6 45.2 33-4 •  Blue v i o l e t Blue v i o l e t Violet Violet V i o l e t red V i o l e t red Violet Blue v i o l e t  o.33 0.53 ().66 1 .32 2.55 4.00 5.30 7.80 8.50 0.50 3 .20  36.1 48.1 49^6 50.6 50.1 46.5 43.7 36.2 33.2 28.8 13.1  Blue Red Red Red Red V i o l e t Violet Violet V i o l e t blue Blue Blue Blue  P Brom. e t h y l a c e t a t e  Glycerol Monochlorhydrin  17  COMPOUND COBTROL Ethylene Chlorohydrin  CONG . «t  Mgs. GUgO  .00  33-8  Blue  0 .62  36.2 38.7 39.5 39.5 35*6 ^5.1 29.7 23.9 19-4 13 .2 33-9 35-2  Blue Blue v i o l e t Violet V i o l et Violet Violet V i o l e t blue Blue Blue Blue  GO  1 .24  2.48  Potassium Thiocvanate  Potassium N i t r i t e  Potassium Chloride  3 .71 4.95 5.60 6.20 7 .44 8.66 9.60 0.01 O.05 0.10 38.4 41 .8 0.20 0.50 42.3 41 .8 1.00 1.50 38.4 2.00 §5.6 2.50 32.1 3.00 30.2 3.60 25.9 0.005 39o2 0.01 40 .0 0.05 48.7 0.10 50.3 0.25 50.9 0.50 52.1 1.00 52.5 2.00 52.9 3.00 54.0 0.00025 37.8 0.0005 40.5 0.001  0.005 0.01 0.02 0.05 0.50 1.00 2.00 6.00 10.00 15.00 20.00 25.00  43.1  53-1 51.7 52.4 54.1  54.4  55.8 55.8 60.0 54.2 41 .0 38.1 35-6  Color Reaction  Blue Blue Blue v i o l e t Blue v i o l e t Violet Blue v i o l e t Violet B l u e v i o l et Blue v i o l e t Blue v i o l e t Blue v i o l e t V i o l e t blue Violet Red v i o l e t Red v i o l e t Red Red Red Red Red Blue v i o l e t Blue v i o l e t Blue v i o l e t Violet Red v i o l e t Red v i o l e t Red Red Red Red Red Red v i o l e t Blue v i o l e t Blue Blue  AMYLASE A C T I V I T Y i n M i l l i g r a m s INHIBITION< > ACCELERATION .  Pigure  1.  Cuprous Oxide.  o  OJ VJ1  o  4V  o  VJl  18. Part I I I The e f f e c t o f c e r t a i n c h e m i c a l s found t o i n c r e a s e amylase a c t i v i t y , upon dormant Dotato tubers . -, m In o r d e r t o examine more c l o s e l y t h e p o s s i b l e r e l a t i o n s h i p .^..between enzyme a c t i v i t y and dormancy i n p l a n t s ,  certain  compounds found t o be most a c t i v e i n t h e s t i m u l a t i o n o f malt d i a s t a s e i n t h e experiments d e s c r i b e d above, were f u r t h e r empl o y e d i n a s e r i e s o f t e s t s upon dormant p o t a t o t u b e r s . In t h e case o f t h e n o n - v o l a t i l e compounds, p a r t i c u l a r l y the  i n o r g a n i c s a l t s , t h e soak method d e s c r i b e d by Denny ( 2 )  was used e x c l u s i v e l y ,  w i t h t h e m o r e v v o l a t i l e o r g a n i c compounds  b o t h t h e soak and t h e d i p methods were employed.  As p l a n t  m a t e r i a l , C e r t i f i e d Seed P o t a t o e s o f t h e ITetted Gem and Up-toDate v a r i e t i e s , harvested, i n mid-October and v a r y i n g i n weight from 4 t o 6 ounces, were used. B e f o r e t r e a t m e n t , t h e t u b e r s were f i r s t washed t o remove all  adhering s o i l .  The s e l e c t e d t u b e r s were then cut l o n g i t -  u d i n a l l y from stem t o seed end i n t o seed p i e c e s o f approximately  2 ounces  i n weight.  T h i s method o f c u t t i n g was used i n  o r d e r t o a v o i d any p o s s i b l e v a r i a t i o n s due t o t h e more r a p i d g e r m i n a t i o n o f t h e seed end. F i v e seed p i e c e s o f each v a r i e t y from as many i n d i v i d u a l t u b e r s , were s e l e c t e d f o r t r e a t m e n t .  I n t h e soak treatment,  t h e s e were soaked i n a p p r o x i m a t e l y one l i t r e o f a 1.5 % s o l u t ion  of the s a l t ,  i n t h e case o f t h e i n o r g a n i c compounds and  a l a n i n e , f o r a p e r i o d o f one hour a t 21 °C.  A f t e r treatment  19» the ing,  seed p i e c e s were removed, d r a i n e d and p l a n t e d w i t h o u t r i n s i n sandy loam i n f l a t s a t a depth of 1-g- i n c h e s .  These  were l e f t under greenhouse c o n d i t i o n s f o r the d u r a t i o n of the experiment, w a t e r b e i n g added to keep the s o i l  sufficiently  moist. In the d i p t r e a t m e n t , seed p i e c e s prepared as above were c o m p l e t e l y immersed i n a 1.0 % s o l u t i o n of the c h e m i c a l under e x a m i n a t i o n and shaken f o r one minute, a f t e r which the excess l i q u i d was d r a i n e d o f f , t h e j a r s s e a l e d t i g h t l y and l e f t a t a t e m p e r a t u r e o f 2 5° f o r 24 h o u r s .  At t h e end of t h i s t i m e , t h e  seed p i e c e s were p l a n t e d i n greenhouse  flats.  C o n t r o l s were p r e p a r e d f o r b o t h the d i p and soak t r e a t e d t u b e r s , u s i n g i n p l a c e o f the d i f f e r e n t s o l u t i o n s ,  distilled  water. A f t e r a p e r i o d o f ?Q days from t h e date o f p l a n t i n g , a comparison i n t h e r e l a t i v e amount of growth from t r e a t e d t u b e r s and c o n t r o l s , was made. cut  F o r t h i s purpose, the p l a n t tops were  o f f a t s o i l l e v e l and t h e green weight of the tops taken  as an i n d e x o f growth.  Results The v a l u e s r e c o r d e d i n T a b l e I I I a r e t h e t o t a l g r e e n weights  of a l l growth from the f i v e seed p i e c e s i n c l u d e d i n each  treatment.  As an a d d i t i o n a l means of comparison, the number of  t u b e r s g e r m i n a t e d out o f the f i v e ,  i s r e c o r d e d i n each case*  The e f f e c t o f c e r t a i n Amylase s t i m u l a t i n g compounds upon t h e g e r m i n a t i o n o f dormant p o t a t o tubers. COMPOUND  NUMBER GERMINATED  GREEN WEIGHT OP TOPS - GRAMS'  NETTED GEE -VARIETY (Dip T r e a t m e n t ! Control Ethyl Acetate / Bromoethyl A c e t a t e G l y c e r o l Monoehlorhydrin 3  2 2 5 2  (Soak Treatment) Control 3 Ethyl Acetate 3 /3 Bromo e t h y l A c e t a t e 5 G l y c e r o l Monoehlorhydrin 2 Alanine 4 P o t a s s i u m Bromide 2 Potassium Chloride 4 Potassium Thiocyanate 5 Potassium. N i t r a t e 4 Potassium N i t r i t e 3  44.0 1 .0 210.0  14.7  27.4 94.2 7333;  75.2 10.2  53.0  104.<  76.3 38.7  UP-TO-DATE VARIETY (Dip Treatment) Control ft Bromo e t h y l A c e t a t e G l y c e r o l Monoehlorhydrin ( Soak Control /3 Bromo e t h y l A c e t a t e Alanine G l y c e r o l Monoehlorhydrin P o t a s s i u m Bromide Potassium Chloride Potassium N i t r a t e Potassium N i t r i t e  2 4 3  Treatment) 1 2 4  3 3  4  43.2  75.3 97.0 40.2  97.1 61 .0  71.2 74.4  151.3  2.4  6,5  Discussion From the d a t a p r e s e n t e d  i n Table I, i t may  be seen  how  d i v e r s e a r e t h e e f f e c t s o f compounds o f v a r i o u s t y p e s upon the a c t i v i t y o f m a l t amylase. d i a s t a t i c a c t i v i t y , these inct classes:  compounds may  compounds h a v i n g  compounds h a v i n g little  On the b a s i s of t h e i r e f f e c t s upon be p l a c e d i n t h r e e  dist-  a definitely accelerating effect;  an i n h i b i t o r y a c t i o n ;  and  compounds e x e r t i n g  influence in either direction. T h i s c l a s s i f i c a t i o n however, i s s u b j e c t t o one  very  import-  ant r e s t r i c t i o n , - t h e c o n c e n t r a t i o n employed, a f a c t o r which appa r e n t l y many workers have n e g l e c t e d . ults listed,  I t i s e v i d e n t from the r e s -  t h a t the e f f e c t produced by a c h e m i c a l  compound i s  l a r g e l y dependant upon i t s c o n c e n t r a t i o n i n the r e a c t i o n , m i x t u r e . Thus, many compounds a r e a c c e l e r a t i n g a t one i n h i b i t i n g at  concentration  but  another.  "Most a c c e l e r a t o r s e x h i b i t e d t h e i r maximum e f f e c t between d e f i n i t e l i m i t s o f c o n c e n t r a t i o n ; on e i t h e r s i d e of t h i s the enzyme  a c t i v i t y may  represented  be a p p r e c i a b l y l e s s .  T h i s optimum e f f e c t i s  g r a p h i c a l l y i n F i g u r e s 1 and 2.  C e r t a i n of the comp-  ounds s t u d i e d however, such as P o t a s s i u m C h l o r i d e and were d e f i n i t e l y s t i m u l a t i n g a t a l l c o n c e n t r a t i o n s O t h e r s were e i t h e r s l i g h t l y a c c e l e r a t i n g o r had  Alanine,  employed.  l i t t l e e f f e c t at  l o w e r c o n c e n t r a t i o n s , but became i n c r e a s i n g l y i n h i b i t o r y v.with the higher concentrations. and  amides.  Of t h i s t y p e were the a l c o h o l s  S t i l l o t h e r s , such as maltose, e t h y l e n e g l y c o l  a c e t o n i t r i l e , had  little  e f f e c t a t any  and  concentration.  With regard to s p e c i f i c i o n s or groups, a few g e n e r a l i z a t -  22 .  i o n s may be drawn. Of t h e i n o r g a n i c compounds, t h o s e c o n t a i n i n g h a l i d e a n i o n s were by f a r t h e most a c t i v e . i n Table I, i t i s evident reased w i t h the increase  In a d d i t i o n , from t h e v a l u e s  given  t h a t t h e s t i m u l a t i n g p r o p e r t i e s deci n atomic weight, o f t h e h a l o g e n .  The  c h l o r i d e i o n was s t r o n g l y a c c e l e r a t i n g v/hile t h e i o d i d e was definitely  inhibiting,  even a t low  concentrations.  A s i m i l a r v a r i a t i o n i n a c t i v i t y was d i s p l a y e d by the orga n i c h a l o g e n compounds.  G l y c e r o l m o n o c h l o r h y d r i n and  ethylene  c h l o r o h y d r i n g r e a t l y i n c r e a s e d t h e d i a s t a t i c a c t i v i t y w h i l e the e t h y l i o d i d e produced an a p p r e c i a b l e in concentrations  decrease.  below 1.0% had l i t t l e  Ethyl  acetate  e f f e c t but on t h e subst-  i t u t i o n o f bromine, became s t r o n g l y a c c e l e r a t i n g , even below  At h i g h  concentrations  became i n h i b i t i n g .  however,  a l l these l a t t e r  The predominance o f o t h e r more  compounds  unfavorable  p r o p e r t i e s masks t h e e f f e c t o f t h e h a l o g e n c o n s t i t u t e n t . may a l s o be t h e fiase w i t h c h l o r o f o r m , ethylene,  d i c h l o r e t h y l ether, d i c h l o r -  e p i c h l o r h y d r i n and c h l o r a l h y d r a t e ,  o b s e r v e d t o produce e i t h e r l i t t l e  This  a l l o f which were  effect or a d e f i n i t e inhibit ion.  In view o f t h e h i g h degree o f a c c e l e r a t i o n brought about by t h e c h l o r i d e i o n , i t seems p r o b a b l e t h e r e f o r e , t h a t t h e a b i l i t y of t h e c h l o r o - o r g a n i c is  compounds t o s t i m u l a t e d i a s t a t i c  due t o the same f a c t o r .  activity,  O r g a n i c compounds o f t h i s type  would u n d o u b t e d l y d i s s o c i a t e s u f f i c i e n t l y t o l i b e r a t e t h e extremely low concentration  o f c h l o r i d e ions r e q u i r e d .  e n t r a t i o n o f 0.0005/2 KC1 e x e r t s an a p p r e c i a b l e r a t e of starch h y d r o l y s i s .  Even a conc-  e f f e c t upon t h e  23. T h i s marked e f f e c t of i n o r g a n i c c h l o r i d e s has  long been  known and many i n v e s t i g a t o r s c l a i m t h a t t r a c e s of such a n i o n s a r e e s s e n t i a l f o r the enzymatic h y d r o l y s i s of s t a r c h . and  D a v i s o n (21)  d e s c r i b e the c h l o r i d e i o n as a 'co-enzyme* o r  specific activator.  Chrempinska (23)  reports that chlorides  produce a d e f i n i t e i n c r e a s e i n d i a s t a t i c a c t i v i t y , a t pH's  Waksman  above the optimum range f o r the enzyme.  particularly  At pH  values  below the optimum,a d e c r e a s e i s produced. I n c o n s i d e r a t i o n of the i n v e s t i g a t i o n s c i t e d above the r e s u l t s p r e s e n t e d h e r e i n , the e x p l a n a t i o n of the data ed by Denny (14)  record-  i n a r e c e n t paper, i s e x t r e m e l y d i f f i c u l t . Only  i n t h e c a s e of p a n c r e a t i n do h i s r e s u l t s i n d i c a t e any i n amylase a c t i v i t y upon the a d d i t i o n of N a d mixture.  and  increase  to the r e a c t i o n  T h i s i s t r u e even when the enzyme s o l u t i o n had  been  p r e v i o u s l y d i a l y s e d , i n which case the a d d i t i o n , of an e l e c t r o l y t e effect.(7)•  would be expected to produce an even more marked Moreover, Denny s t a t e s t h a t i n no case was  any  increase i n  enzyme a c t i v i t y observed upon the a d d i t i o n of e t h y l e n e rin.  This  chlorohyd-  i s a f u r t h e r d i v e r g e n c e from the r e s u l t s recorded  t h i s present  paper and  in  i n t h a t p r e v i o u s l y p u b l i s h e d by C l a r k et  a l ( 1 ) . In Denny's experiments however, the h y d r o l y s i s was  allow-  ed t o c o n t i n u e  hours.  f o r the r e l a t i v e l y l o n g p e r i o d of eighteen  I t seems l i k e l y t h e r e f o r e , t h a t h i s f a i l u r e to o b t a i n an -increase i n amylase a c t i v i t y i n the p r e s e n c e of NaCl and h y d r i n , may c o n t r o l s and  be due  chloro-  t o t h e f a c t t h a t t h e h y d r o l y s i s b o t h i n the  i n t h e m i x t u r e s c o n t a i n i n g the c h e m i c a l ,  reached the e q u i l i b r i u m p o i n t . ing  ethylene  had  almost  I t i s p r o b a b l e t h a t any a c c e l e r a t -  e f f e c t would be more apparent i n the e a r l y stages of the r e a c t -  24.  ion,  t h a n toward the end. In t h e second p a r t o f t h e l a t t e r paper, Denny s t a t e s t h a t  r e l a t i v e l y l a r g e amounts o f HaCI were added to t h e r e a c t i o n mixt u r e i n o r d e r t o m i n i m i z e the i n f l u e n c e o f s m a l l t r a c e s o f c h l orides present i n t h e ethylene  chlorhydrin.  However, i n view  o f t h e f a c t t h a t t h e s t i m u l a t i n g e f f e c t of e t h y l e n e and  chlorohydrin  o t h e r s i m i l a r compounds, upon malt d i a s t a s e , i s l i k e l y due  to t h e i r s l i g h t d i s s o c i a t i o n i n t o c h l o r i d e i o n s , t h e a d d i t i o n of Ha CI  would d e f e a t  t h e o b j e c t o f t h e experiment.  The a c t i v i t y o f  the c h l o r o h y d r i n would be e n t i r e l y masked by t h e p r e s e n c e o f the ]>7aCl.  I n a d d i t i o n , t h e l a t t e r compound, w i t h i t s h i g h  a t i o n , would a p p r e c i a b l y  ioniz-  decrease the d i s s o c i a t i o n of the chloro-  h y d r i n by t h e common i o n e f f e c t . Of t h e o t h e r  i n o r g a n i c a n i o n s s t u d i e d , the s u l f a t e and  t h e n i t r a t e had l i t t l e of malt diastase.  e f f e c t upon t h e a m y l o c l a s t i c  properties  P o t a s s i u m n i t r i t e however, was almost as  e f f i c i e n t an a c c e l e r a t o r as t h e c h l o r i d e . A l a n i n e and g l y c i n e were found t o be d e f i n i t e l y a c c e l e r a t i n g i n t h e i r a c t i o n and t y r o s i n e o n l y s l i g h t l y so, a l t h o u g h its  l o w s o l u b i l i t y prevented i t s use i n h i g h e r  concentrations.  Acetamide and t h e o t h e r amides recorded exerted i n f l u e n c e i n the lower concentrations i t i n g at higher  concentrations.  little  but were a p p r e c i a b l y i n h i b -  These o b s e r v a t i o n s  including  amino a c i d s and amides a r e i n accordance w i t h the p r e v i o u s l y c i t e d work of Rockwood ( 8 ) . Urea and p h e n y l urea had l i t t l e ion. pointed  effect in either direct-  I t i s apparent t h e r e f o r e , as Sherman and H a y l o r ( i i ) have out, t h a t t h e presence o f t h e HH2 group i n a compound i s  25o  i n s u f f i c i e n t to produce enzymatic ' a c t i v a t i o n ' . the molecule i s of f i r s t  I t sposition i n  importance.  On replacement o f t h e oxygen o f t h e urea compounds, by s u l f u r , t h e r e s u l t i n g compounds such as t h i o u r e a , p h e n y l t h i o u r e a , and t o l y l t h i o u r e a , became d e f i n i t e l y a c c e l e r a t i n g .  Methyl  urea was observed t o have a s i m i l a r e f f e c t , but whether t h i s i s due t o t h e p r e s e n c e o f b t h e m e t h y l group,  i s uncertain.  P o t a s s i u m t h i o c y a n a t e was found t o be d e f i n i t e l y ating  i n i t s a c t i o n upon m a l t d i a s t a s e by b o t h t h e Wohlgemuth  and t h e copper r e d u c t i o n methods. lished  stimul-  C o n t r a r y to the r e s u l t s pub-  i n t h e p r e v i o u s paper by C l a r k , F o w l e r and B l a c k (1), conc-  e n t r a t i o n s above 2.0$ KSCH were a p p r e c i a b l y i n h i b i t i n g .  Maximum  a c c e l e r a t i o n , by t h e copper r e d u c t i o n method appeared t o be a t approximately 0  w h i l e by t h e i o d i n e method, i t was n e a r e r  1.0$.  Both Denny (>!4) and M i l l e r (18) r e p o r t t h a t t h e a c c e l e r a t i o n i s apparent o n l y i n t h e a l k a l i n e r e a c t i o n range. a c i d i c c o n d i t i o n s , i n h i b i t i o n i s produced. do t h o s e i n T a b l e I I , 2 . i s  Under  Denny's r e s u l t s , as  i n d i c a t e t h a t a c o n c e n t r a t i o n o f KSCH o f  d e f i n i t e l y i n h i b i t o r y a t a pH o f 6.4.  However, had Denny  c a r r i e d out h i s experiments o v e r a wider range of c o n c e n t r a t i o n s he would no doubt have o b t a i n e d an a p p r e c i a b l e a c c e l e r a t i o n w i t h t h e KSCN a t even l o w e r pH v a l u e s . Hydroxy ase a c t i v i t 3  r  compounds i n g e n e r a l were found to decrease amyl-  to a g r e a t e x t e n t .  I n t h i s r e s p e c t , members of t h e  a r o m a t i c s e r i e s were c o n s i d e r a b l y more a c t i v e than those o f t h e aliphatic series.  M e t h y l and e t h y l a l c o h o l s were i n h i b i t i n g at  c o n c e n t r a t i o n s above 8.0$ w h i l e phenol, c r e s o l , hydroqulnone and p h l o r o g l u c i h o l were e q u a l l y e f f e c t i v e a t c o n c e n t r a t i o n s of 0«3$  26.  Waksman and Davison  (21) a t t r i b u t e t h i s p r o p e r t y o f a l c o h o l  to t h e i r e f f e c t upon t h e degree o f d i s s o c i a t i o n o f t h e enzyme and t o t h e i n f l u e n c e upon t h e c o l l o i d a l s t a t e "both o f t h e enzyme andrthe  substrate.  I t would seem e v i d e n t t h e r e f o r e , t h a t the  i n h i b i t i o n induced by t h e h i g h e r c o n c e n t r a t i o n s o f e t h y l e n e c h l o r o h y d r i n i s due t o a s i m i l a r cause, s i n c e t h e c h l o r o h y d r i n and e t h y l a l c o h o l a r e such c l o s e l y r e l a t e d  compounds.  A q u a l i t a t i v e comparison o f t h e data o b t a i n e d by t h e Wohlgemuth and t h e copper r e d u c t i o n methods o f d e t e r m i n i n g  diastatic  a c t i v i t y , y i e l d s no o u t s t a n d i n g p o i n t s o f d i f f e r e n c e .  I n Table  II,  o n l y i n t h e case o f t h e t h r e e h i g h e s t c o n c e n t r a t i o n s o f K S C T  a r e c o l o i s observed data.  which do hot correspond  The c o l o r s noted,  t o t h e r e d u c i n g sugar  i n comparison w i t h t h e b l u e o f t h e con-  t r o l , would suggest t h a t the h y d r o l y s i s had proceeded to a p o i n t f u r t h e r i n advance o f t h a t i n d i c a t e d by the accompanying copper reduction values.  S i m i l a r o b s e r v a t i o n s were made by Johnson and  Wormall ( 2 4 ) , who thus concluded  t h a t t h e t h i o c y a n a t e exerted i t s  a c c e l e r a t i n g e f f e c t only i n t h e e a r l i e r stages of h y d r o l y s i s . I t i s p o s s i b l e however, t h a t t h e d i s c r e p a n c i e s observed may be due to the e f f e c t o f t h e t h i o c y a n a t e upon t h e s t a r c h - i o d i n e c o l o r r e a c t i o n , r a t h e r than t h e p r e f e r e n t i a l s t i m u l a t i o n o f the amylo c l a s t i c a c t i v i t y o f t h e enzyme.  The data o b t a i n e d i s i n s u f f i c -  i e n t t o draw any a c c u r a t e c o n c l u s i o n s r e g a r d i n g t h i s aspect o f the p r o b l e m . Curve 1. i n F i g u r e 2. i l l u s t r a t e s c l e a r l y , t h e s e n s i t i v e ness o f amylase p r e p a r a t i o n s t o extremely low c o n c e n t r a t i o n s of chlorides.  The r e l a t i v e l y h i g h e r c o n c e n t r a t i o n s o f t h e c h l o r o -  o r g a n i c compounds r e q u i r e d t o produce an e q u i v a l e n t e f f e c t , i s  27.  no doubt due to t h e i r much l o w e r d i s s o c i a t i o n , as w e l l as t h e p a r a l l e l i n h i b i t o r y e f f e c t e x e r t e d by o t h e r c o n s t i t u t e n t s o f the molecule.  A l l compounds of t h i s t y p e show a f a i r l y wide  o p t i m a l c o n c e n t r a t i o n range.  /$ Bromoethyl  a c e t a t e however,  e x e r t s i t s maximum e f f e c t o n l y between v e r y narrow l i m i t s . In F i g u r e 1. the e f f e c t of KSCN and K N 0 obscure.  They may,  2  i s r a t h e r more  l i k e the c h l o r i d e s , have some i n f l u e n c e  i n a s s i s t i n g i n t h e d i s s o c i a t i o n o f t h e enzyme molecule  i n t o the  a c t i v e c a t i o n , b e l i e v e d t o be d i r e c t l y r e s p o n s i b l e i n s t a r c h hydrolysis. While the d a t a p r e s e n t e d i n Table I I I cannot be c o n s i d e r ed s i g n i f i c a n t , from a s t a t i s t i c a l s t a n d p o i n t owing t o the r e l a t i v e l y s m a l l numbers of t u b e r s used i n the v a r i o u s t r e a t m e n t s , they do a s s i s t i n drawing f u r t h e r c o n c l u s i o n s r e g a r d i n g the r e l a t i o n between enzyme a c t i v i t y and dormancy. In most cases, treatment of the dormant t u b e r s w i t h those c h e m i c a l s found t o e x e r t a marked i n f l u e n c e upon malt d i a s t a s e , d i d r e s u l t i n much e a r l i e r g e r m i n a t i o n o f the t r e a t e d t u b e r s and i n i n c r e a s e d growth by the end of the  experiment.  The two v a r i e t i e s of p o t a t o e s used i n t h e s e t e s t s , showed c e r t a i n v a r i a t i o n s with, r e s p e c t to t h e d i f f e r e n t With seed p i e c e s o f t h e Netted Gem  chemicals.  v a r i e t y , (5  bromoethyl  a c e t a t e was by f a r the most e f f e c t i v e , both i n the number o f germinated  seed p i e c e s and in. t h e t o t a l amount of growth  The next most e f f e c t i v e was KSCN.  A l a n i n e , KC1,  produced,  and KNO^ a l l  induced g e r m i n a t i o n i n f o u r out of t h e o r i g i n a l f i v e seed p i e c e s . G l y c e r o l Monochlorohydrin  was  e q u a l l y e f f e c t i v e w i t h r e s p e c t to  the g r e e n weight of t h e t o p s but caused g e r m i n a t i o n i n o n l y two  28.  of the treated pieces. t h e enzyme, had l i t t l e  KKOg w h i l e h a v i n g a marked e f f e c t upon e f f e c t upon t h e dormant t u b e r s .  Using t h e Up-to-Date v a r i e t y , KC1 was most e f f e c t i v e . Glyc e r o l m o n o c h l o r o h y d r i n and ft bromoethyl a c e t a t e were s l i g h t l y l e s s e f f i c i e n t t h a n t h e c h l o r i d e , but w e r e ' e q u i v a l e n t i n t h e i r effect.  K B r and a l a n i n e , as w i t h t h e N e t t e d Gem t u b e r s ,  a l s o capable of breaking  were  t h e dormancy.  Thus, w i t h t h e e x c e p t i o n  o f KN0 , a l l compounds found to 2  s t i m u l a t e t h e a c t i v i t y o f m a l t d i a s t a s e , produced a corresponding  response i n dormant t u b e r s . In view o f t h e f a i l u r e o f c h e m i c a l s ,  a c t i v e i n breaking  known t o be extremely  dormancy, t o produce a s i m i l a r i n c r e a s e i n  t h e amylase a c t i v i t y o f p o t a t o  j u i c e , Denny concludes t h a t the  a c t i o n o f t h e s e compounds i s e s s e n t i a l l y i n d i r e c t i n n a t u r e . As f u r t h e r e v i d e n c e f o r t h i s c o n c l u s i o n , he has shown t h a t chlorohydrin, while having l i t t l e  i n f l u e n c e upon t h e d i a s t a t i c  a c t i v i t y when added d i r e c t l y to t h e expressed p o t a t o e x h i b i t an a p p r e c i a b l e  ethylene  juice, did  e f f e c t when t h e t u b e r s were f i r s t  treated  w i t h t h e c h e m i c a l and t h e j u i c e expressed a f t e r s e v e r a l days. I n a d d i t i o n to t h i s , Denny has been u n a b l e t o d u p l i c a t e the r e s u l t s of C l a r k e£ a l ( l ) w i t h e t h y l e n e therefore, that h i s previous  c h l o r o h y d r i n and KSCN and c l a i m s  conclusions  r e q u i r e no m o d i f i c a t i o n .  As the w r i t e r s of t h e l a t t e r paper have p o i n t e d out, the f a c t t h a t t h e s e compounds have l i t t l e of the potato ity  e f f e c t upon t h e a c t i v i t y  j u i c e , does not n e c e s s a r i l y p r e c l u d e  the p o s s i b i l -  t h a t a d i r e c t as w e l l as an i n d i r e c t e f f e c t may e x i s t . I t  may be p o s s i b l e £hat t h e f a i l u r e t o o b t a i n any i n c r e a s e i n amyl-  29.  ase a c t i v i t y i s due  r a t h e r to the p a r a l l e l a c t i v a t i o n of antag-  o n i s t i c enzymes o r t o s i d e r e a c t i o n s o f the chemicals w i t h o t h e r c o n s t i t u e n t s o f the p o t a t o As Denny suggests, breaking  chemicals  and  employed  juice.  the r e l a t i o n s h i p between dormancy-  the v a r i o u s enzyme systems of l i v i n g  i s u n d o u b t e d l y very complex.  ceils  Moreover, the a b i l i t y of a comp-  ound t o induce a renewal of growth i n dormant p l a n t s must c e r t a i n l y be defendant upon a v a r i e t y of f a c t o r s . That t h e a c t i v a t i o n of t h e amylase o f r e s t i n g p l a n t s i s one  important  f u n c t i o n at l e a s t , of t h e chemicals  inducing germination, cribed.  capable of  i s obvious from the experiments h e r e i n des-  Under p r o p e r c o n d i t i o n s of c o n c e n t r a t i o n and r e a c t i o n ,  t h o s e compounds most e f f i c i e n t i n b r e a k i n g  t h e r e s t p e r i o d of  p l a n t s , b r i n g about a d e f i n i t e i n c r e a s e i n the a c t i v i t y of malt amylase. That a r e l a t i o n s h i p of some k i n d e x i s t s between the d i s t i n c t p r o p e r t i e s of t h e chemic a l s  i n question,  d i c a t e d by t h e r e s u l t s t a b u l a t e d i n T a b l e I I I .  two  i s further inAlanine,  Glycer-  o l m o n o c h l o r h y d r i n and (2> bromoethyl a c e t a t e , h i t h e r t o u n t r i e d i n t h e i r e f f e c t upon dormant p l a n t s , were found to be e q u a l l y as eff«o i c i e n t i n t h a t r e s p e c t as the e t h y l e n e r e p o r t e d by Denny.  These chemicals  c h l o r o h y d r i n and KSCN f i r s t  v/ere s e l e c t e d f o r the germ-  i n a t i o n t e s t s on the b a s i s of t h e i r a b i l i t y to s t i m u l a t e malt diastase. c h e m icals  I t seems apparent t h e r e f o r e , t h a t ' the e f f e c t of i n inducing sprouting  i s more d i r e c t i n some phases at  l e a s t , than Denny's c o n c l u s i o n s would i n d i c a t e . r  The f o r e g o i n g r e s u l t s and t h e i r d i s c u s s i o n have y i e l d e d l i t t l e i n f o r m a t i o n o f v a l u e concerning  the mechanism of enzyme  30.  r e a c t i o n s and  t h e i r r e l a t i o n , to dormancy i n p l a n t s .  hoped however, t h a t the d a t a p r e s e n t e d may suggesting  It is  prove of value  f u r t h e r methods o f approach t o t h e s e and  in  related  problems.  SUMMAHY  1.  The  e f f e c t of a number o f o r g a n i c and  in varying concentrations,  i n o r g a n i c compounds  upon the a m y l o c l a s t i c and  saccharog-  e n i c a c t i v i t y of malt amylase, has been r e c o r d e d . 2.  On the b a s i s of t h e i r i n f l u e n c e upon amylase a c t i v i t y ,  the c h e m i c a l s s t u d i e d have been c l a s s i f i e d a s : a c c e l e r a t o r s , i n h i b i t o r s , or compounds h a v i n g l i t t l e e f f e c t .  Whether a comp-  ound i s a c c e l e r a t i n g o r i n h i b i t i n g i n n a t u r e has been found to be l a r g e l y dependant upon i t s c o n c e n t r a t i o n  i n the r e a c t i o n med-  ium. 3«  The h i g h , degree of s t i m u l a t i o n noted i n t h e case o f  i n o r g a n i c c h l o r i d e s , has  l e d to the s u g g e s t i o n  e f f e c t exerted by c h l o r o - o r g a n i c  the  t h a t the s i m i l a r  compounds i s due  to t h e i r p a r t -  i a l d i s s o c i a t i o n , w i t h the l i b e r a t i o n of c h l o r i d e i o n s . 4.  Of t h e n i t r o g e n compounds examined, amino a c i d s e x h i b i t e d  the g r e a t e s t a c c e l e r a t i o n . in t h e i r action.  Amides were a p p r e c i a b l y  Urea compounds were e f f e c t i v e a c c e l e r a t o r s  o n l y when t h e oxygen was ponding thio-compound.  r e p l a c e d by s u l f u r , f o r m i n g the P o t a s s i u m n i t r i t e was  u l a t i n g i n c h a r a c t e r , w h i l e t h e n i t r a t e was 5.  Ho  inhibiting  e v i d e n c e was  obtained  corres-  exceedingly  only s l i g h t l y  stimso*  which would suggest t h a t any  of  31. t h e compounds s t i m u l a t e d p r e f e r e n t i a l l y e i t h e r the a m y l o c l a s t i c o r t h e s a c c h a r o g e n i c phases o f s t a r c h h y d r o l y s i s . 60  E t h y l e n e c h l o r o h y d r i n and P o t a s s i u m t h i o c y a n a t e , compounds  known t o be e f f e c t i v e i n b r e a k i n g t h e r e s t p e r i o d o f p l a n t s , have been shown t o be a c c e l e r a t i n g i n t h e i r e f f e c t upon malt diastase.  T h i s d a t a i s p r e s e n t e d as evidence o f the p o s s i b l e  r e l a t i o n s h i p between amylase a c t i v i t y and the b r e a k i n g o f dormancy e 7.  E a r l i e r g e r m i n a t i o n and growth o f dormant p o t a t o t u b e r s  has been brought  about by treatment w i t h c e r t a i n  chemicals  p r e v i o u s l y found to e x e r t a s t i m u l a t i n g e f f e c t upon t h e a c t i v i t y o f malt d i a s t a s e .  T h i s d a t a i s p r e s e n t e d as f u r t h e r e v i d -  ence o f a d i r e c t r e l a t i o n between the a b i l i t y o f a compound to i n d u c e g e r m i n a t i o n and  i t s e f f e c t upon t h e p l a n t amylase*  * # a- *  32.  BIBLIOGRAPHY Clark,  R.H,, Fowler, F.L.,Black, P e t e r T», T r a n s . Roy. S o c . Canada. Sec.3. 25: 99, Denny, P .E. Am. J o u r n . o f B o t . 13; 386, 1926. Denny, E.E. J o u r n . I n d . & Eng. 'Chem. 20:578, 1928.  1931.  Denny, F.E. C o n t r i h . Boyce Thompson I n s t . 3:277, 1931. Denny, F.E. C o n t r i b , Boyce Thompson I n s t . 4:53, 1932. Nasse. Arch. ges. P h y s i o l .  9: 138,  1875.  Preti. Biochem. Z.  4:1,  1907.  Rockwood. J o u r n . Am. Chem. Soc. Effront.  Hon. S c i .  39:2745, 1917.  41:266, 1893•  Sherman and Walker. J o u r n . Am. Chem. Soc. Sherman and N a y l o r . J o u r n . Am. Chem. Soc. Sherman and C a l d w e l l . J o u r n . Am. Chem. Soc.  43:2461, 1921. 44:2957, 1922. 44:2924, 1922.  Euler. Fermentforschung. Denny, F.E.  3:330, 1920.  C o n t r i b . Boyce Thompson I n s t . Wohlgemuth, J . C i t e d by Waksman and Davison, (see r e f . 21 .) Ohlsson. Soc. B i o l . 87:1183, 1922.  5:441,  1933•  "Enzymes',  1  P»159«  33«  1? • 18. 19. 20.  C l a r k , ¥7.LI. "The D e t e r m i n a t i o n o f Hydrogen Ions." p. 2 1 4 . 1923 e d i t i o n . M i l l e r , L.P. C o n t r i b . Boyce Thompson I n s t . 3 : 2 8 7 , 1 9 3 1 . Sherman and Wayman. J o u r n . Am. Chem. S o c .  43*2456,  S c h u l t z and L a n d i s . J o u r n . Am. Chem. S o c .  54:211,  1921.  1932.  21 •  Waksman and D a v i s o n . "Enzymes" W i l l i a m s and W i l k i n s ,  22.  O f f i c i a l and T e n t a t i v e Methods o f A n a l y s i s o f t h e A s s o c i a t i o n o f O f f i c i a l A g r i c u l t u r a l Chemists, Wash.  23.  Chrempinska,  Baltimore, 1 9 2 6 .  Hedwiga.  Biochem. J o u r n .  25:  Ho.5.,  1931•  24.  Johnson, L.R. and Wormall, A. P r o c . Leeds P h i l . & L i t . S o c , S c i . Sec.1 :318, 1928. C i t e d by L.P. M i l l e r . ( 1 8 )  25.  Waldschmidt and Walton. "Enzyme a c t i o n s and P r o p e r t i e s " . John W y l i e & Sons. 1929.  NOTE:  R e f e r e n c e s marked  (*) c i t e d i n ( 2 5 ) .  

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