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The influence of certain chemicals upon amylase activity Edwards, Howard I. 1934-10-31

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1 - ! j ACC. P*0. &£2?&Q^„^^ f THE INFLUENCE OE CERTAIN CHEMICALS UPON AMYLASE ACTIVITY by Howard I . Edwards *#«« A Thesis submitted f o r the Degree of MASTER OP ARTS i n the Department of CHEMISTRY * * «• The U n i v e r s i t y of B r i t i s h Columbia A p r i l , 1934. ACKNOWLEDGMENT , The w r i t e r wishes to express h i s thanks to Dr. R.H. Clark, Head of the Department of Chemistry, under whose d i r e c t i o n t h i s research was c a r r i e d out, f o r the advice given and the i n t e r e s t shown during the progress of the v/ork; to Dr. A.H, Hutchinson, Head of the Department of Botany who so k i n d l y pro vided the greenhouse space necessary i n the germin a t i o n experiments; and to Mr. H.S. McLeod, D i s t r i c t Inspector, Dominion Department of A g r i c u l t u r e , who supplied the C e r t i f i e d Seed Potatoes used i n these experiments• # # # * « TABLE OP CONTENTS INTRODUCTION Previous work upon Dormancy i n p l a n t s . Review of the 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 of methods of measurement a v a i l a b l e . PART I The a m y l o c l a s t i c a c t i v i t y of malt d i a s t a s e . Experimental Methods Results Table I PART I I The eaccharogenic a c t i v i t y of malt d i a s t a s e . Experimental Methods Res u l t s Table I I Figure 1 . Figure 2 . PART I I I The e f f e c t of c e r t a i n chemicals upon dormant potato tubers. Experimental Methods Res u l t s Table I I I DISCUSSION SUMMARY BIBLIOGRAPHY 1. THE INFLUENCE OE CERTAIN CHEMICALS UPON AMYLASE ACTIVITY. ** **»*»*«• In t r o d u c t i o n In a p r e l i m i n a r y report of 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 , Clark, Fowler and Black (1) have shown that potassium thiocyanate, ethylene c h l o r o h y d r i n and th 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 the a c t i v i t y of malt d i a s ~ t a s e . From the data obtained, i t was suggested that the a b i l  i t y of these compounds to induce a renewal of growth i n dormant plants Awas due probably to t h e i r a c t i o n upon the enzymes concer ned i n the u t i l i z a t i o n of the food required f o r the i n i t i a l growth and germination, and more p a r t i c u l a r l y , the pla n t amyl ases o In t h i s report and i n a more recent paper by Denny ( 4 ) , i t i s stated however, that no increase i n amylase a c t i v i t y was observed when such chemicals were added d i r e c t l y to the f r e s h l y expressed potato j u i c e . From t h i s , Denny concludes that the e f f e c t of the thiocyanate 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 nature 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 of the potato amylase. In support of t h i s conclusion, Denny f u r t h e r shows (5) that ethylene c h l o r o h y d r i n while having no appreciable e f f e c t when added to the expressed j u i c e d i r e c t l y , d i d produce a def i n i t e increase i n amylase a c t i v i t y when the 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 e f f e c t only i n the case of l e s s dormant potatoes. He suggests that the power of such compounds to 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 the p l a n t c e l l s i n t o the formation of a greater am ount of enzyme, ra t h e r than the s t i m u l a t i o n of that already pre sent. In a d d i t i o n to the work c i t e d above, a considerable number of 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 other phases of amyl« ase a c t i v i t y . As e a r l y as 1875» Nasse (6) reported that there was an important and s p e c i f i c dependance i n the a c t i v i t y of f e r  ments, upon the presence of s a l t s , while P r e t i (7) reported that amylases from d i f f e r e n t sources were rendered p r a c t i c a l l y i n e r t by d i a l y s i s , but re s t o r e d to a c t i v i t y by the a d d i t i o n of neut r a l e l e c t r o l y t e s . A l a r g e p r o p o r t i o n of the experiments have been confined to the e f f e c t of amino acids and other p r o t e i n products upon d i a s t a t i c a c t i v i t y . Rockwood (8) has s t u d i e d the e f f e c t of a l a r g e number of 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 that the amino acid s were a c c e l e r a t i n g i n character while amides were not. E f f r o n t (9) a l s o obtained s i m i l a r res u l t s and a s c r i b e d t h i s property to t h e i r amphoteric character 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 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 to be p r o t e i n i n nature, r a p i d l y d e t e r i o r a t  es i n an aqueous s o l u t i o n . This l o s s i n a c t i v i t y i s considered by Sherman and Walker (10) to be due to the h y d r o l y s i s of the enzyme. They suggest that the 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  y s i s . Sherman and Naylor (11) report that benzoic a c i d , hipp- 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 e f f e c t upon the a c t i v i t y of s a l i v a r y and p a n c r e a t i c amylase. Thus, n e i t h e r the presence of the carboxyl and amino groups, alone, or togeth er i n the same molecule, was s u f f i c i e n t to a c c e l e r a t e the enzyme, but only compounds of the amino type were e f f e c t i v e . That there was some t r u t h i n RockwoocTs theory, was demonstrated by Sherman and.Caldwell (12). In t h e i r experiments they had found that amino acids exerted a p r o t e c t i v e i n f l u e n c e against the inh i b i t i o n of p a n c r e a t i c amylase by mercuric c h l o r i d e . ITo explan- of t h i s was o f f e r e d , but i f , as E u l e r suggests (13) such t o x i c - its'" i s caused by the combination of the mercuric ion w i t h the enzyme molecule, the amino aci d s may prevent t h i s by combining w i t h the m e t a l l i c i o n . Up to the present time however, l i t t l e experimental work has been c a r r i e d out i n determining the e f f e c t of s p e c i f i c chem i c a l groups or ions upon amylase a c t i v i t y . Moreover, a review of the 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 comparatively l i t t l e i n f  ormation concerning the e f f e c t of the concentration of the chem i c a l ' a c t i v a t o r s ' , upon the enzyme a c t i v i t y . I t was considered t h e r e f o r e , that 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 concentrations, might prove o f value i n throwing f u r t h e r l i g h t on the mechanism of enzyme react- ions and the r e l a t i o n of amylase a c t i v i t y to dormancy. In add i t i o n , because of the divergence i n r e s u l t s presented i n (1) and i n a recent paper by Denny (14), i t was considered necessary to repeat the experiments w i t h potassium thiocyanate and ethylene c h l o r o h y d r i n and extending them to a wider range of concentrat i o n s . 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 the ab i l i t y of a compound to break the r e s t period of 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 , those compounds found to be most a c c e l e r a t i n g i n character were employed i n an exper- iment to determine t h e i r power i n breaking the dormancy of pot ato tubers. The usual methods of measuring d i a s t a t i c a c t i v i t y f a l l i n t o two general c l a s s e s ; those determining the rate of disappearance of the substrate, and those determining the q u a n t i t y of r e a c t i o n products formed. Of the f i r s t type, that most commonly used i s the Wohlgemuth method (15)» based upon the a l t e r a t i o n i n the c o l  o r r e a c t i o n w i t h iodine, o c c u r r i n g during s t a r c h h y d r o l y s i s . Measurements of t h i s type are an index of the l i q u e f y i n g or amyl- o c l a s t i c power of the enzyme. Of the second type, the usual cop per r e d u c t i o n procedure i s f r e q u e n t l y employed i n determining the r a t e of formation of reducing substances. This i s an index of the saccharogenic power of the enzyme. Whether the r e s u l t s obtained by the two methods are compar able i s s t i l l a matter of some controversy. Most i n v e s t i g a t o r s c l a i m that 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 at l e a  s t two d i s t i n c t enzyme f r a c t i o n s of d i a s t a s e . A6 yet however, a l l attempts to separate the two f r a c t i o n s have been unsuccess f u l . Ohlsson (16) announced that by a b r i e f heating of 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 of the system while yet r e t a i n i n g the l i q u e f y i n g act i o n . I t i s p o s s i b l e that i n the case of amylase, instead, of two d i s t i n c t enzymes, the s i n g l e enzyme molecule may co 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 the degradation of sta r c h to dextri n e and the other h y d r o l y s i n g the d e x t r i n s to red ucing bodies. Of the two methods of measurement, the determination of the a m y l o c l a s t i c power may be the more b a s i c , since before the formation of reducing sugars can occur, i t seems evident that part of the s t a r c h at l e a s t must f i r s t be hydrolysed to the i n t  ermediate d e x t r i n s . In a d d i t i o n to t h i s , the Wohlgemuth method i s much more r a p i d l y executed. Since one of the objects of t h i s i n v e s t i g a t i o n was the t e s t i n g of a l a r g e number of compounds, many of which might render the more complex copper r e d u c t i o n inaccurate or even impossible, i t was decided to employ the form er procedure, modified i n some d e t a i l s , as a measure of amylase a c t i v i t y . However, f o r purposes of comparison with Denny's r e s u l t s (14 :), those 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 using the standard copper reduction procedure. Par t I The Influence of the Concentration of C e r t a i n Chemical Compounds upon the A m y l o c l a s t i c Act i v i t y of Malt D i a s t a s e . Experimental Procedure: As a source of the enzyme, malt d i a s t a s e , U.S.P. IX, prep ared by Eimer and Amend, was used. For-each day's experiments, 1 .0 gram of the enzyme m a t e r i a l was extracted f o r 30 minutes at room temperature w i t h 5°° ccs. 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 residue 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 , through a f o l d e d f i l t e r . As s u b s t r a t e , s o l u b l e s t a r c h (Baker's, C .P.')• was employed, one l o t being used e x c l u s i v e l y throughout the e n t i r e i n v e s t i g a t  i o n . As i n the case of the enzyme e x t r a c t , a f r e s h s o l u t i o n was prepared each day by mixing 15»0 grams of the s t a r c h w i t h 100 ccs d i s t i l l e d water and heating i n a water bath t i l l s o l u t  ion was complete. This was f i n a l l y d i l u t e d to a volume of 200 c c s . The r e a c t i o n mixture c o n s i s t e d of: 7»5 ccs. of 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 ccs. enzyme ex 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 varying proportions of a s o l u t i o n of the chemical under examination. The whole was then made up to a volume of 25*0 ccs. This mixture a f t e r being p l a c ed i n test-tubes and corked, was incubated i n an e l e c t r i c oven at a temperature of 30°C»» f o r the course of the experiment. S t a r t i n g approximately two hours a f t e r the beginning of the experiment, the r a t e of disappearance of s t a r c h was observed by withdrawing a few drops of the r e a c t i o n mixture from time to time and t e s t i n g w i t h N/200 Iodine 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 time required f o r the h y d r o l y s i s to reach the ach romic po i n t , or the point at which no c o l o r other than that of the i o d i n e was apparent, was noted i n each case. S u f f i c i e n t i o d i n e was added to 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 , a l t h  ough care was taken i n i t s a d d i t i o n , f o r an excess obscured the end point • As Denny (14) and M i l l e r (18) have pointed out, c e r t a i n compounds, p a r t i c u l a r l y potassium thiocyanate and t h i o u r e a react with the iodine and are l i k e l y to render the s t a r c h - i o d i n e t e s t i n a c c u r a t e . In the case of the thiocyanate i t was found that by adding 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 mixture 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 under 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 necessary to use a l a r g e r volume of the r e a c t i o n mixt 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 with the t h i o u r e a before the s t a r c h r e a c t i o n could be ob served . Even under these circumstances, i t was d i f f i c u l t to o b t a i n accurate readings. The q u a n t i t y of enzyme employed was s u f f i c i e n t to c a r r y the h y d r o l y s i s to the achromic point i n approximately f o u r hours. I t was considered more s a t i s f a c t o r y to complete each run i n a s i n g l e day, i n order to avoid the use of p r e s e r v a t i v e s such as toluene, which, apart from i t s i n h i b i t o r y e f f e c t (19) might react w i t h c e r t a i n of the chemicals i n use. P r e l i m i n a r y experiments al s o i n d i c a t e d that constant shaking, p a r t i c u l a r l y when toluene was added, decreased the r a t e of h y d r o l y s i s appreciably, (appoint noted by S c h u l t z and Landis (20) i n t h e i r experiments with 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 glassware a f t e r c l e a n i n g with chromic a c i d , thoroughly r i n s e d with d i s t i l l e d water before use. A l l pH measurements were made wit h the quinhydrone electr o d e i n a l l but a few instances when the c o l o r i m e t r i c method was used. Compounds having too great 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. presence of the b u f f e r , were omitted from f u r t h e r t e s t s . P r e l i m i n a r y experiments i n d i c a t e d that f o r t h i s sample of malt d i a s t a s e at l e a s t , the optimum r e a c t i o n range l a y between pH 6.0 and pK 6 . 5 , although the optimum range u s u a l l y given i s pK 4.0 - 4.5 (21) . I t i s q u i t e probablp however, that 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 temperature. (21) . The b u f f e r mixture used, was adjusted toa pH of 6.2. 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 the r e a c t i o n mixture, a PH of 6.4 f i n a l l y r e s u l t e d . Ho pH v a r i a t i o n s were g r e a t e r than ±.15• #**«*# R e s u l t s The values redorded i n Table I represent the decrease (acc 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 presence of the chem i c a l s l i s t e d , as compared to the c o n t r o l s . The average time r e q u i r e d f o r the appearance of the achromia poin t i n the c o n t r o l tubes was approximately 245 minutes. 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 balanced by comparing each day's experiments w i t h the c o n t r o l s f o r that day o n l y . A l l values presented are the mean of se v e r a l readings. In cases where i n h i b i t i n g compounds had so delayed the r e a c t i o n that i t was impossible to o b t a i n a value f o r the achromic point that day, the minimum degree of i n h i b i t i o n i s recorded. This was done only where h y d r o l y s i s was apparent, as evidenced by the red or purple c o l o r a t i o n of the intermediate d e x t r i n s with i o d i n e . In cases where the compound was so s t r o n g l y i n h i b i t i n g that no d e x t r i n s could be detected by means of the iodine t e s t , but only the blue c o l o r of the s t a r c h was apparent, the e f f e c t i s record 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 the presence of c e r t a i n chemical compounds. COMPOUND Potassium Bromide Potassium Chloride Potassium Iodide Potassium N i t r i t e Potassium N i t r a t e Potassium S u l f a t e CONC. ACCEL. INK IB % (Minutes) 0.01 0.03 100 0.10 121 1.0 120 0.0004 0 0.001 35 0.004 80 0.01 98 0.03 115 0.10 133 1.0 140 2.0 150 0.01 14 0.03 18 0.10 63 •2.0 180 0.005 35 0.01 55 0.05 87 0.20 102 0.40 120 2.0 137 3.0 125 0.01 30 0.03 41 0.10 34 0.60 15 0.01 0 0.03 0 0.10 2 0.60 13 1.0 12 10 COMPOUND CQNC • ACCEL. INHIB. _% (Minutes). Potassium Thiocyanate 0.10 37 0.50 80 O.75 90 1.0 100 1.5 43 2.0 9 3300 38 Ethylene Chiorohydrin 0.24 17 0.60 38 1.20 55 2.48 71 3.72 80 4.96 80 6.20 53 6.80 7 7.44 78 E p i c h l o r h y d r i n 0.80 2 1.6 16 2.4 42 G l y c e r o l MoBichlorhydrin 0.05 31 (Symmet.) 0.22 72 O.65 96 * .0 120 1 .64 1322 • G l y c e r o l ffionbehlbrhydrin 0.26 79 1.32 122 4.0 149 5.30 88 8.0 32 C h l o r a l Hydrate 0.20 0 1.0 8 2.0 10 D i c h l o r e t h y l e n e 0.025 0 0.10 0 .0375 2 D i c h l o r e t h y l ether 0.23 23 0.46 74 1.20 >130 Chloroform 0.06 6 0.27 0 0 0.90 39 11. COMPOUND COHC . -HCSt ^ B K l AG GEL. INHIB E t h y l Iodide 0.10 3 0.20 11 0.30 21 E t h y l Bromide 0.218 9 0.58 12 1 .16 16 1 .45 22 n. P r o p y l Bromide 0.27 6 0.54 22 1 .36 10 Ethylene G l y c o l 0 .44 0 0 .90 5 2.23 7 Propylene G l y c o l 0 .41 0 0.84 2 2.10 2 3.10 12 Trimethylene G l y c o l 0.21 1 0 .42 10 1 .02 14 2.05 22 3.10 29 Methyl A l c o h o l 0.47 5 1 .60 3 6.38 34 15.90 160 E t h y l A l c o h o l 0.80 1 3.16 6 9.50 13 15.8 120 A l l y l A l c o h o l O.56 8 1 .13 15 3 .50 52 6.90 > 120 17.10 > 120 Acetone 0.31 0 0.80 20 1.58 30 3.16 23 6.32 2 9.50 12 15.80 135 COMPOUND 12. CONC. ACCEL. 1KB" IB Maltose 0.20 15 0.50 14 1.00 11 2.00 5 3-GO 30 Glucose 0.50 0 1.0 5 2.0 10 3.0 34 E t h y l Acetate 0.90 2 1 .80 5 3-58 21 7.20 >120 0 Brom E t h y l Acetate 0.30 58 0.90 53 1 .50 47 3.0 10 A l a n i n e 0.08 40 0.24 77 0.40 91 1.20 118 Glyc ine 0.10 19 0.50 24 1100 40 Tyrosine 0.004 2 0.02 10 0.03 20 Acetamide 0.08 9 0.20 13 0.40 16 0.60 16 0.80 27 1 .20 46 Propionamide 0.08 1 0.40 6 0.80 9 1 .20 as •Butyramide 0.04 0 0 0.08 0 0.20 15 0.60 21 1.20 62 13- COMPOUND CONC. ACCEL Oxamide 0 .005 3 0.010 8 0 .0375 26 Urea 0.10 11 0.50 3 1.00 1.50 Thiourea 0.10 20 0.50 48 1.00 77 2.00 63 Phenyl Urea 0.015 0 0.04.5 6 0.225 25 Phenyl Thiourea 0.005 2 0.020 12 0 .05 23 0.075 37 T o l y l Thiourea 0.01 12 0.10 38 Methyl Urea 0.16 26 0 .40 39 1 .20 56 Creatine Hydrate 0.04 0 0.20 10 0 ,60 24 P y r i d i n e 0.20 12 0 .80 5.90 Com] P i p e r i d i n e 0.070 0.17 P y r r o l 0.01 16 0 .06 22 0 .09 27 0.17 30 Succ inimide 0 .02 0 0.08 4 0.30 20 INHIB. 9 20 85 24 140 1 4 . COMPOUND SONC:W. 0.16 0.60 1.60 2.40 ACCEL . INK IB. A c e t o n i t r i l e 0 0 8 15 Phenol 0 . 1 0 0.20 0.60 1.00 12 0 130 225 o. Chlorphenol 0 . 0 1 2 0.062 0 . 1 2 4 0.31 0 . 6 2 0 .93 Complete « 3 6 15 100 I r i h i b . « p. C r e s o l 0.10 0 .40 0.60 0.80 4 0 80 > 1 2 0 > 1 2 0 Hydroquinone 0.10 0.50 1.00 Complete 38 > 1 ? 0 Inh i b . P h i o r o g l u c i n o l 0 . 0 4 0 . 2 0 o .6§ 17 100 > 2 0 0 P a r t I I The i n f l u e n c e of c e r t a i n chemicals upon the saccharogenic a c t i v i t y of Malt D i a s t a s e . Experimental Procedure The method employed d i f f e r e d from that described above only i n decreasing the q u a n t i t y of enzyme extract added to the 15- r e a c t i o n mixture, from 1.5 toO.75 c c s . The tubes containing the h y d r o l y s i n g mixture were incubated at 30°C« f o r a p e r i o d of four hours, r a t h e r than the longer p e r i o d of eighteen hours used-by Denny (14) f o r the reason t h a t any a c c e l e r a t i o n due to the add- ed chemicals would be more evident i n the e a r l y stages of hyd r o l y s i s than when the r e a c t i o n had almost reached e q u i l i b r i u m . P r e l i m i n a r y experiments i n d i c a t e d that t h i s was a c t u a l ^ the case. At the end of the f o u r hour period, 5.0 cc. volumes of the r e a c t i o n mixture were p i p e t t e d into 20.0 ccs. Fehling's s o l u t i o n and the reducing sugars determined by the standard Munson and Walker procedure (22) . Cuprous oxide was determined by the volumetric permanganate method. Prom the cuprous oxide values thus obtained were deducted those from blank determinat ions obtained by the same procedure except that 0.75 ccs. b o i l e d enzyme s o l u t i o n were added. I t was found impossible to apply t h i s method to mixtures cont a i n i n g t h i o u r e a however, owing to the decomposition of the l a t t e r compound on heating w i t h Fehling's s o l u t i o n with the form a t i o n of a copper s u l f i d e . Attempts to use the c o l o r i m e t r i c p i c  r i c a c i d method were eq u a l l y u n s a t i s f a c t o r y . The values obtained are presented below. Re s u l t s In Table 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 of cuprous oxide. Each value given i s the mean of s e v e r a l determinations. As a f u r t h e r means of comparison with the Wohlgemuth method, the col o r s noted on the a d d i t i o n of iodine 16. to a few drops of the r e a c t i o n mixture at the end of the four hour p e r i o d , are recorded. The e f f e c t of the concentration of c e r t a i n of the compounds upon the a c t i v i t y of the enzyme, i s represented g r a p h i c a l l y i n Figures 1 and 2 » COMPOUND TABLE I I CONG. /o Mgs. Cu 2 0 Color Reaction CONTROL Alanin e P Brom. e t h y l acetate G l y c e r o l Monochlorhydrin 00 0.10 0.20 0.^0 1 .00 1.50 1.80 3.6o 0.015 0.06 0.15 0.30 0.45 0.60 1.20 1.50 o.33 0.53 ().66 1 .32 2.55 4.00 5.30 7.80 8.50 0.50 3 .20 13.8 Blue 36.1 Blue 38.9 Blue v i o l e t 44.5 V i o l e t 51.8 V i o l e t 52.9 Red v i o l e t 53.6 V i o l e t red 56 .4 Red 38.5 Blue v i o l e t 40.4 Blue v i o l e t 42 .2 V i o l e t 46.5 V i o l e t 50.2 V i o l e t red 49.6 V i o l e t red 45.2 V i o l e t 33-4 • Blue v i o l e t 36.1 Blue 48.1 Red 49^6 Red 50.6 Red 50.1 Red V i o l e t 46.5 V i o l e t 43.7 V i o l e t 36.2 V i o l e t blue 33.2 Blue 28.8 Blue 13.1 Blue 17 COMPOUND COBTROL Ethylene Chlorohydrin Potassium Thiocvanate Potassium N i t r i t e Potassium Chloride CONG . «t Mgs. GUgO Color Reaction GO .00 33-8 Blue 0 .62 36.2 Blue 1 .24 38.7 Blue v i o l e t 2.48 39.5 V i o l e t 3 .71 39.5 V i o l et 4.95 35*6 V i o l e t 5.60 ^5.1 V i o l e t 6.20 29.7 V i o l e t blue 7 .44 23.9 Blue 8.66 19-4 Blue 9.60 13 .2 Blue 0.01 33-9 Blue O.05 35-2 Blue 0.10 38.4 Blue v i o l e t 0.20 41 .8 Blue v i o l e t 0.50 42.3 V i o l e t 1.00 41 .8 Blue v i o l e t 1.50 38.4 V i o l e t 2.00 §5.6 Blue v i o l et 2.50 32.1 Blue v i o l e t 3.00 30.2 Blue v i o l e t 3.60 25.9 Blue v i o l e t 0.005 39o2 V i o l e t blue 0.01 40 .0 V i o l e t 0.05 48.7 Red v i o l e t 0.10 50.3 Red v i o l e t 0.25 50.9 Red 0.50 52.1 Red 1.00 52.5 Red 2.00 52.9 Red 3.00 54.0 Red 0.00025 37.8 Blue v i o l e t 0.0005 40.5 Blue v i o l e t 0.001 43 . 1 Blue v i o l e t 0.005 53-1 V i o l e t 0.01 51.7 Red v i o l e t 0.02 52.4 Red v i o l e t 0.05 54.1 Red 0.50 54.4 Red 1.00 55.8 Red 2.00 55.8 Red 6.00 60.0 Red 10.00 54.2 Red v i o l e t 15.00 41 .0 Blue v i o l e t 20.00 38.1 Blue 25.00 35-6 Blue AMYLASE A C T I V I T Y i n M i l l i g r a m s C u p r o u s O x i d e . I N H I B I T I O N < > ACCELERATION . P i g u r e 1 . o O J V J 1 o 4V o VJl 18. Part I I I The e f f e c t of c e r t a i n chemicals found to inc  rease amylase a c t i v i t y , upon dormant Dotato tubers . -, m In order to examine more c l o s e l y the p o s s i b l e r e l a t i o n - ship .^ ..between enzyme a c t i v i t y and dormancy i n p l a n t s , c e r t a i n compounds found to be most a c t i v e i n the s t i m u l a t i o n of malt d i a s t a s e i n the experiments described above, were f u r t h e r emp loye d i n a s e r i e s of t e s t s upon dormant potato tubers. In the case of the 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 norganic s a l t s , the soak method described by Denny ( 2 ) was used e x c l u s i v e l y , with the m o r e v v o l a t i l e organic compounds both the soak and the dip methods were employed. As plant m a t e r i a l , C e r t i f i e d Seed Potatoes of the ITetted Gem and Up-to- Date v a r i e t i e s , harvested, i n mid-October and varying i n weight from 4 to 6 ounces, were used. Before treatment, the tubers were f i r s t washed to remove a l l adhering s o i l . The selected tubers were then cut l o n g i t  u d i n a l l y from stem to seed end into seed pieces of approximat e l y 2 ounces i n weight. This method of c u t t i n g was used i n order to avoid any p o s s i b l e v a r i a t i o n s due to the more rapid germination of the seed end. F i v e seed pieces of each v a r i e t y from as many i n d i v i d u a l tubers, were s e l e c t e d f o r treatment. In the soak treatment, these were soaked i n approximately one l i t r e of a 1.5 % solut ion of the s a l t , i n the case of the inorganic compounds and al a n i n e , f o r a per i o d of one hour at 21 °C. A f t e r treatment 19» the seed pieces were removed, drained and planted without r i n s  ing, i n sandy loam i n f l a t s at a depth of 1-g- inches. 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, water being added to keep the s o i l s u f f i c i e n t l y m o ist. In the dip treatment, seed pieces prepared as above were completely immersed i n a 1.0 % s o l u t i o n of the chemical under examination and shaken f o r one minute, a f t e r which the excess l i q u i d was drained o f f , the j a r s sealed t i g h t l y and l e f t at a temperature of 2 5° f o r 24 hours. At the end of t h i s time, the seed pieces were planted i n greenhouse f l a t s . C ontrols were prepared f o r both the dip and soak treated tubers, using i n place of the d i f f e r e n t s o l u t i o n s , d i s t i l l e d water. A f t e r a p e r i o d of ?Q days from the date of p l a n t i n g , a comparison i n the r e l a t i v e amount of growth from t r e a t e d tubers and c o n t r o l s , was made. For t h i s purpose, the pl a n t tops were cut o f f at s o i l l e v e l and the green weight of the tops taken as an index of growth. Results The values recorded i n Table I I I are the t o t a l green weig ht s of a l l growth from the f i v e seed pieces included i n each treatment. As an a d d i t i o n a l means of comparison, the number of tubers germinated out of the f i v e , i s recorded i n each case* The e f f e c t of c e r t a i n Amylase s t i m u l a t i n g comp ounds upon the germination of dormant potato t u b e r s . COMPOUND NUMBER GERMINATED NETTED GEE -VARIETY (Dip Treatment! GREEN WEIGHT OP TOPS - GRAMS' C o n t r o l 2 E t h y l Acetate 2 /3 Bromoethyl Acetate 5 G l y c e r o l Monoehlorhydrin 2 (Soak Treatment) C o n t r o l 3 E t h y l Acetate 3 /3 Bromo e t h y l Acetate 5 G l y c e r o l Monoehlorhydrin 2 A l a n i n e 4 Bromide 2 C h l o r i d e 4 Thiocyanate 5 N i t r a t e 4 N i t r i t e 3 Potassium Potassium Potassium Potassium. Potassium UP-TO-DATE VARIETY (Dip Treatment) Co n t r o l ft Bromo e t h y l Acetate G l y c e r o l Monoehlorhydrin ( Soak Control /3 Bromo e t h y l Acetate Ala n i n e G l y c e r o l Monoehlorhydrin Potassium Bromide Potassium C h l o r i d e 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 44.0 1 .0 2 1 0 . 0 14.7 27.4 94 .2 7 3 3 3 ; 75.2 1 0 . 2 53.0 104.< 76.3 38.7 43.2 75.3 97.0 40.2 97.1 61 .0 71.2 74.4 151.3 2.4 6,5 D i s c u s s i o n From the data presented i n Table I, i t may be seen how d i v e r s e are the e f f e c t s of compounds of various types upon the a c t i v i t y of malt amylase. On the b a s i s of t h e i r e f f e c t s upon d i a s t a t i c a c t i v i t y , these compounds may be placed i n three d i s t  i n c t c l a s s e s : compounds having a d e f i n i t e l y a c c e l e r a t i n g e f f e c t ; compounds having an i n h i b i t o r y a c t i o n ; and compounds exerting l i t t l e i n f l u e n c e i n e i t h e r d i r e c t i o n . This c l a s s i f i c a t i o n however, i s subject to one very import ant r e s t r i c t i o n , - the concentration employed, a f a c t o r which app a r e n t l y many workers have neglected. I t i s evident from the res u l t s l i s t e d , that the e f f e c t produced by a chemical compound i s l a r g e l y dependant upon i t s concentration i n the reaction, mixture. Thus, many compounds are a c c e l e r a t i n g at one concentration but i n h i b i t i n g at 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 of c o n c e n t r a t i o n ; on e i t h e r side of t h i s the enz yme a c t i v i t y may be appreciably l e s s . This optimum e f f e c t i s represented g r a p h i c a l l y i n Figures 1 and 2. C e r t a i n of the comp ounds stud i e d however, such as Potassium Chloride and Alanine, were d e f i n i t e l y s t i m u l a t i n g at a l l concentrations employed. Others 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 or had l i t t l e e f f e c t at lower concentrations, 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. Of t h i s t y p e were the a l c o h o l s and amides. S t i l l others, such as maltose, ethylene g l y c o l and a c e t o n i t r i l e , had l i t t l e e f f e c t at any concentration. With regard to s p e c i f i c ions or groups, a few g e n e r a l i z a t -22 . ions may be drawn. Of the inorganic compounds, those containing h a l i d e anions were by f a r the most a c t i v e . In a d d i t i o n , from the values given i n Table I, i t i s evident that the s t i m u l a t i n g p r o p e r t i e s dec reased w i t h the increase i n atomic weight, of the halogen. The c h l o r i d e ion was s t r o n g l y a c c e l e r a t i n g v/hile the iodide was def i n i t e l y i n h i b i t i n g , even at 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 displayed by the org anic halogen compounds. G l y c e r o l monochlorhydrin and ethylene c h l o r o h y d r i n g r e a t l y increased the d i a s t a t i c a c t i v i t y while the e t h y l i o d i d e produced an appreciable decrease. E t h y l acetate i n concentrations below 1.0% had l i t t l e e f f e c t but on the subst i t u t i o n of 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 however, a l l these l a t t e r compounds became i n h i b i t i n g . The predominance of other more unfavorable p r o p e r t i e s masks the e f f e c t of the halogen c o n s t i t u t e n t . This may a l s o be the fiase w i t h chloroform, d i c h l o r e t h y l ether, d i c h l o r - ethylene, e p i c h l o r h y d r i n and c h l o r a l hydrate, a l l of which were observed to produce e i t h e r l i t t l e e f f e c t or a d e f i n i t e i n h i b i t i o n . In view of the high degree of a c c e l e r a t i o n brought about by the c h l o r i d e ion, i t seems probable therefore, that the a b i l i t y of the chloro-organic compounds to stimulate d i a s t a t i c a c t i v i t y , i s due to the same f a c t o r . Organic compounds of t h i s type would undoubtedly d i s s o c i a t e s u f f i c i e n t l y to l i b e r a t e the extrem ely low concen t r a t i o n of c h l o r i d e ions r e q u i r e d . Even a conc e n t r a t i o n of 0.0005/2 KC1 exerts an appreciable e f f e c t upon the r a t e of st a r c h h y d r o l y s i s . 23. This 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 claim that traces of such anions are 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 . Waksman and Davison (21) describe the c h l o r i d e ion as a 'co-enzyme* or s p e c i f i c a c t i v a t o r . Chrempinska (23) reports that c h l o r i d e s produce a d e f i n i t e increase i n d i a s t a t i c a c t i v i t y , p a r t i c u l a r l y at pH's above the optimum range f o r the enzyme. At pH values below the optimum,a decrease i s produced. In 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 and the r e s u l t s presented h e r e i n , the explanation of the data record ed by Denny (14) i n a recent paper, i s extremely d i f f i c u l t . Only i n the case 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 increase 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 to the r e a c t i o n mixture. This 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 addition, of an e l e c t r o l y t e would be expected to produce an even more marked e f f e c t . ( 7 ) • Moreover, Denny sta t e s that 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 ethylene chlorohyd r i n . This i s a f u r t h e r divergence from the r e s u l t s recorded i n t h i s present paper and i n that p r e v i o u s l y published by Clark et a l ( 1 ) . In Denny's experiments however, the h y d r o l y s i s was allow ed to continue f o r the r e l a t i v e l y long period of eighteen hours. I t seems l i k e l y t h e r e f o r e , that h i s f a i l u r e to obtain an -increase i n amylase a c t i v i t y i n the presence of NaCl and ethylene chloro h y d r i n , may be due to the f a c t t h a t the h y d r o l y s i s both i n the c o n t r o l s and i n the mixtures con t a i n i n g the chemical, had almost reached the e q u i l i b r i u m p o i n t . I t i s probable that any a c c e l e r a t  ing e f f e c t would be more apparent i n the e a r l y stages of the react-2 4 . ion, than toward the end. In the second part of the l a t t e r paper, Denny states that r e l a t i v e l y l a r g e amounts of HaCI were added to the r e a c t i o n mix tu r e i n order to minimize the i n f l u e n c e of small traces of c h l  o r i d e s present i n the ethylene c h l o r h y d r i n . However, i n view of the f a c t that the s t i m u l a t i n g e f f e c t of ethylene c h l o r o h y d r i n and other 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 ions, the a d d i t i o n of Ha CI would defeat the object of the experiment. The a c t i v i t y of 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 the presence of the ]>7aCl. In a d d i t i o n , the l a t t e r compound, with i t s high i o n i z  a t i o n , would ap p r e c i a b l y decrease the d i s s o c i a t i o n of the chloro h y d r i n by the common ion e f f e c t . Of the other inorganic anions studied, the s u l f a t e and the n i t r a t e had l i t t l e e f f e c t upon the a m y l o c l a s t i c p r o p e r t i e s of malt d i a s t a s e . Potassium 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 the c h l o r i d e . Alanine and g l y c i n e were found to 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 only s l i g h t l y so, although i t s low s o l u b i l i t y prevented i t s use i n higher concentrations. Acetamide and the other amides recorded exerted l i t t l e i n f l u e n c e i n the lower concentrations but were appreciably i n h i b  i t i n g at higher concentrations. These observations i n c l u d i n g amino acid s and amides are 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 phenyl urea had l i t t l e e f f e c t i n e i t h e r d i r e c t  i o n . I t i s apparent th e r e f o r e , as Sherman and Haylor ( i i ) have pointed out, that the presence of the 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 ' . I t s p o s i t i o n i n the molecule i s of f i r s t importance. On replacement of the oxygen of the urea compounds, by s u l f u r , the r e s u l t i n g compounds such as thiou r e a , phenyl thiourea, 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 to have a s i m i l a r e f f e c t , but whether t h i s i s due to the presence ofbthe methyl group, i s u n c e r t a i n . Potassium thiocyanate was found to be d e f i n i t e l y s t i m u l  a t i n g i n i t s a c t i o n upon malt d i a s t a s e by both the Wohlgemuth and the copper re d u c t i o n methods. Contrary to the r e s u l t s pub l i s h e d i n the previous paper by Clark, Fowler and Black (1), conc e n t r a t i o n s above 2.0$ KSCH were ap 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 the copper r e d u c t i o n method appeared t o be at approximately 0 w h i l e by the iod i n e method, i t was nearer 1.0$. Both Denny (>!4) and M i l l e r (18) report that the ac c e l e r  a t i o n i s apparent o n l y i n the a l k a l i n e r e a c t i o n range. Under a c i d i c c o n d i t i o n s , i n h i b i t i o n i s produced. Denny's r e s u l t s , as do those i n Table I I , i n d i c a t e that a concentration of KSCH of 2 . i s d e f i n i t e l y i n h i b i t o r y at a pH of 6.4. However, had Denny c a r r i e d out h i s experiments over a wider range of concentrations he would no doubt have obtained an appreciable a c c e l e r a t i o n with the KSCN at even lower pH v a l u e s . Hydroxy compounds i n general were found to decrease amyl ase a c t i v i t 3 r to a great extent. In t h i s respect, members of the aromatic s e r i e s were considerably more a c t i v e than those of the a l i p h a t i c s e r i e s . Methyl and ethyl a l c o h o l s were i n h i b i t i n g at concentrations above 8.0$ 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 equally e f f e c t i v e at concentrations of 0«3$ 26. Waksman and Davison (21) a t t r i b u t e t h i s property of a l c o h o l to t h e i r e f f e c t upon the degree of d i s s o c i a t i o n of the enzyme and to the i n f l u e n c e upon the c o l l o i d a l s t a t e "both of the enzyme andrthe s u b s t r a t e . I t would seem evident therefore, that the i n h i b i t i o n induced by the higher concentrations of ethylene c h l o r ohydrin i s due to a s i m i l a r cause, since the chlor o h y d r i n and et h y l a l c o h o l are 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 of the data obtained by the Wohl gemuth and the copper re d u c t i o n methods of determining d i a s t a t i c a c t i v i t y , y i e l d s no outstanding points of d i f f e r e n c e . In Table I I , o n l y i n the case of the three highest concentrations of KSCT are c o l o i s observed which do hot correspond to the reducing sugar data. The c o l o r s noted, i n comparison with the blue of the con t r o l , would suggest that the h y d r o l y s i s had proceeded to a point f u r t h e r i n advance of that i n d i c a t e d by the accompanying copper red u c t i o n values. S i m i l a r observations were made by Johnson and Wormall (24), who thus concluded that the thiocyanate 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 the 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, that the discrepancies observed may be due to the e f f e c t of the thiocyanate upon the st 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 the p r e f e r e n t i a l s t i m u l a t i o n of the amyl- o c l a s t i c a c t i v i t y of the enzyme. The data obtained i s i n s u f f i c  i e n t to draw any accurate conclusions regarding t h i s aspect of the problem. Curve 1. i n Fi g u r e 2. i l l u s t r a t e s c l e a r l y , the s e n s i t i v e  ness of amylase preparations to extremely low concentrations of c h l o r i d e s . The r e l a t i v e l y higher concentrations of the chloro- organic compounds required to produce an equivalent e f f e c t , i s 27. no doubt due to t h e i r much lower d i s s o c i a t i o n , as w e l l as the p a r a l l e l i n h i b i t o r y e f f e c t exerted by other c o n s t i t u t e n t s of the molecule. A l l compounds of t h i s type show a f a i r l y wide op t i m a l c o n c e n t r a t i o n range. /$ Bromoethyl acetate however, exerts i t s maximum e f f e c t only between very narrow l i m i t s . In Figure 1. the e f f e c t of KSCN and KN0 2 i s rather more obscure. They may, l i k e the c h l o r i d e s , have some in f l u e n c e i n a s s i s t i n g i n the d i s s o c i a t i o n of the enzyme molecule into the a c t i v e c a t i o n , b e l i e v e d to be d i r e c t l y r e s p o n s i b l e i n starch hyd r o l y s i s . While the data presented i n Table I I I cannot be consider 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 standpoint owing to the r e l  a t i v e l y small numbers of tubers used i n the various treatments, they do a s s i s t i n drawing f u r t h e r conclusions regarding 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 tubers with those chemicals found to exert a marked i n f l u e n c e upon malt diastase, di d r e s u l t i n much e a r l i e r germination of the treated tubers and i n increased growth by the end of the experiment. The two v a r i e t i e s of potatoes used i n these t e s t s , showed c e r t a i n v a r i a t i o n s with, respect to the d i f f e r e n t chemicals. With seed pieces of the Netted Gem v a r i e t y , (5 bromoethyl acetate was by f a r the most e f f e c t i v e , both i n the number of ger minated seed pieces and in. the t o t a l amount of growth produced, The next most e f f e c t i v e was KSCN. Alanine, KC1, and KNO^ a l l induced germination i n four out of the 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 equally e f f e c t i v e with respect to the green weight of the tops but caused germination i n only two 28. of the treated p i e c e s . KKOg while having a marked e f f e c t upon the enzyme, had l i t t l e e f f e c t upon the dormant tub e r s . Using the Up-to-Date v a r i e t y , KC1 was most e f f e c t i v e . Gly c e r o l monochlorohydrin and ft bromoethyl acetate were s l i g h t l y l e s s e f f i c i e n t than the c h l o r i d e , but were'equivalent i n t h e i r e f f e c t . KBr and a l a n i n e , as with the Netted Gem tubers, were a l s o capable of breaking the dormancy. Thus, w i t h the exception of KN0 2, a l l compounds found to s t i m u l a t e the a c t i v i t y of malt d i a s t a s e , produced a correspond ing response i n dormant tub e r s . In view of the f a i l u r e of chemicals, known to be extremely a c t i v e i n breaking dormancy, to produce a s i m i l a r increase i n the amylase a c t i v i t y of potato j u i c e , Denny concludes that the a c t i o n of these compounds i s e s s e n t i a l l y i n d i r e c t i n nature. As f u r t h e r evidence f o r t h i s conclusion, he has shown that ethylene c h l o r o h y d r i n , while having l i t t l e i n f l u e n c e upon the 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 the expressed potato j u i c e , d i d e x h i b i t an ap p r e c i a b l e e f f e c t when the tubers were f i r s t t r e a ted w i t h the chemical and the j u i c e expressed a f t e r s e v e r a l days. In a d d i t i o n to t h i s , Denny has been unable to d u p l i c a t e the r e s u l t s of Clark e£ a l ( l ) with ethylene chlorohydrin and KSCN and claims t h e r e f o r e , t h a t h i s previous conclusions require no m o d i f i c a t i o n . As the w r i t e r s of the l a t t e r paper have pointed out, the f a c t that these compounds have l i t t l e e f f e c t upon the a c t i v i t y of the potato j u i c e , does not n e c e s s a r i l y preclude the p o s s i b i l  i t y that 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 . It may be p o s s i b l e £hat the f a i l u r e to obtain any increase i n amyl-29. ase a c t i v i t y i s due rather 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 or to s i d e r e a c t i o n s of the chemicals employed wit h other c o n s t i t u e n t s of the potato j u i c e . As Denny suggests, the r e l a t i o n s h i p between dormancy- breaking chemicals and the various enzyme systems of l i v i n g c e i l s i s undoubtedly very complex. Moreover, the a b i l i t y of a comp ound to 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 the a c t i v a t i o n of the amylase of r e s t i n g plants i s one important f u n c t i o n at l e a s t , of the chemicals capable of inducing germination, i s obvious from the experiments her e i n des c r i b e d . Under proper c o n d i t i o n s of concentration and r e a c t i o n , those compounds most e f f i c i e n t i n breaking the r e s t period of p l a n t s , b r i n g about a d e f i n i t e increase 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 two d i s t i n c t p r o p e r t i e s of the chemicals i n question, i s f u r t h e r i n  d i c a t e d by the r e s u l t s tabulated i n Table I I I . Alanine, Glycer o l monochlorhydrin and (2> bromoethyl acetate, 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 equally as eff«o i c i e n t i n t h a t respect as the ethylene c h l o r o h y d r i n and KSCN f i r s t reported by Denny. These chemicals v/ere selec 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 stimulate malt d i a s t a s e . I t seems apparent therefore, that' the e f f e c t of chemicals 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 conclusions would i n d i c a t e . r The foregoing 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 nformation of value 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 . I t i s hoped however, that the data presented may prove of value i n suggesting f u r t h e r methods of approach to these and r e l a t e d problems. SUMMAHY 1. The e f f e c t of a number of organic and inorganic compounds in varying concentrations, upon the a m y l o c l a s t i c and saccharog- enic a c t i v i t y of malt amylase, has been recorded. 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 chemicals studied have been c l a s s i f i e d as: a c c e l e r a t o r s , i n h i b i t o r s , or compounds having 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 or i n h i b i t i n g i n nature has been found to be l a r g e l y dependant upon i t s concentration i n the r e a c t i o n med ium. 3« The high, degree of s t i m u l a t i o n noted i n the case of the inorganic c h l o r i d e s , has l e d to the suggestion that the s i m i l a r e f f e c t exerted by chloro-organic compounds i s due to t h e i r part i a l d i s s o c i a t i o n , with 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 the n i t r o g e n compounds examined, amino acid s e x h i b i t e d the greatest a c c e l e r a t i o n . Amides were appreciably i n h i b i t i n g i n t h e i r a c t i o n . Urea compounds were e f f e c t i v e a c c e l e r a t o r s only when the oxygen was replaced by s u l f u r , forming the corres ponding thio-compound. Potassium n i t r i t e was exceedingly stim u l a t i n g i n character, while the n i t r a t e was only s l i g h t l y so* 5. Ho evidence was obtained which would suggest that any of 31. the compounds stimulated 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 or the saccharogenic phases of starch h y d r o l y s i s . 6 0 Ethylene c h l o r o h y d r i n and Potassium thiocyanate, compounds known to be e f f e c t i v e i n breaking the r e s t period of p l a n t s , have been shown to 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 d i a s t a s e . This data i s presented as evidence of 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 breaking of dorm ancy e 7 . E a r l i e r germination and growth of dormant potato tubers 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 exert a s t i m u l a t i n g e f f e c t upon the a c t i v  i t y of malt d i a s t a s e . This data i s presented as f u r t h e r evid ence of a d i r e c t r e l a t i o n between the a b i l i t y of a compound to induce germination and i t s e f f e c t upon the plant amylase* * # a- * 32. BIBLIOGRAPHY Clark, R.H,, Fowler, F.L.,Black, Peter T», Trans. Roy. Soc. Canada. Sec.3. 25: 99, 1931. Denny, P .E. Am. Journ. of Bot. 13; 386, 1926. Denny, E.E. Journ. Ind. & Eng. 'Chem. 20:578, 1928. Denny, F.E. Co n t r i h . Boyce Thompson In s t . 3:277, 1931. Denny, F.E. Co 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. P r e t i . Biochem. Z. 4:1, 1907. Rockwood. Journ. Am. Chem. Soc. 39:2745, 1917. E f f r o n t . Hon. S c i . 41:266, 1893• Sherman and Walker. Journ. Am. Chem. Soc. 43:2461, 1921. Sherman and Naylor. Journ. Am. Chem. Soc. 44:2957, 1922. Sherman and C a l d w e l l . Journ. Am. Chem. Soc. 44:2924, 1922. E u l e r . Fermentforschung. 3:330, 1920. Denny, F.E. Con t r i b . Boyce Thompson I n s t . 5:441, 1933• Wohlgemuth, J . Cited by Waksman and Davison, "Enzymes',1 P»159« (see r e f . 21 .) Ohlsson. Soc. B i o l . 87:1183, 1922. 33« 1? • Clark, ¥7.LI. "The Determination of Hydrogen Ions." p. 2 1 4 . 1923 e d i t i o n . 18. M i l l e r , L.P. Co n t r i b . Boyce Thompson I n s t . 3 : 2 8 7 , 1 9 3 1 . 1 9 . Sherman and Wayman. Journ. Am. Chem. Soc. 4 3 * 2 4 5 6 , 1 9 2 1 . 2 0 . S chultz and Landi s . Journ. Am. Chem. Soc. 5 4 : 2 1 1 , 1 9 3 2 . 21 • Waksman and Davison. "Enzymes" Williams and W i l k i n s , Baltimore, 1 9 2 6 . 2 2 . O f f i c i a l and Tentative Methods of A n a l y s i s of the A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists, Wash. 2 3 . Chrempinska, Hedwiga. Biochem. Journ. 25: Ho .5 . , 1931• 2 4 . Johnson, L.R. and Wormall, A. Proc. Leeds P h i l . & L i t . S o c , S c i . Sec.1 :318, 1928. Cited by L.P. M i l l e r . ( 1 8 ) 25. Waldschmidt and Walton. "Enzyme ac t i o n s and P r o p e r t i e s " . John Wylie & Sons. 1929. NOTE: References marked (*) c i t e d i n ( 2 5 ) . 

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