Magdalen College, Oxford. (Two Figures in Text.)

THE DIFFERENCES OF ACTION OF VARIOUS DIA-STASES. BY H. M. VERNON, M.A., M.D., Fellow ofMagdalen College, Oxford. (Two Figures in Text.)

(From the Physiological Laboratory, Oxford.)

THE COLOUR REACTIONS WITH IODINE.

WHEN estimating the diastatic action of pancreatic extracts on dilutestarch by means of the achromic point method, it was noticed that therate of progress in the colour reactions with iodine varied considerablywith different extracts. In order to investigate this rate of progressquantitatively, two arbitrary colour standardls were chosen, and theiodine colour reactions of the digesting starch compared with them.The first standard, of a deep violet colour, was a faintly alkalitne solutionof haematoxylin, and the secon(l, of a pale reddish brown, a solution ofpicrocarmine. Such quantities of these stains were taken as to givesolutions which corresponded almost exactly, both in shade and depth,to those obtainable on addition of small quantities of digesting starch todilute iodine solution. Fresh 1 0/0 starch paste made with distilledwater was used, and this was diluted with an equal volume of tap water,less the amount of extract to be added. The test-tubes or flasks ofdigesting starch were kept in a water bath at 38', and small quantitiesof the digest liquid removed fromn time to time by means of a pipette,and run into test-tubes of diltute iodine held at an angle over a sheet ofwhite paper. The times taken by the digest to reach the above-mentioned deep violet and reddish brown stages could thus bedetermined to within less than i minute, they being, in fact, easier tofix than the achromic point time. Such a quantity of extract wasgenerally taken as to give an achromic point time of 10 to 15 minutes.

The results obtained with various extracts are given in the accoin-panying table. Most of the values here given are the means of severalsets of observations, made with different amounts of ferment. Variationsin the quantity of ferment only very slightly affect the relative rate of

ACTION OF DIASTASES.

progress of the colour reactions, hence they may forignored. For instance, in the case of the glycerin

our puirpose beextract of dog's

Extract

500/0 glycerin ext. dog75 0/0 gljycerin ext. pig75 0/ ,. ,, (kept 25 hours)25 0/. alcohol750/0 glycerin ext. woman25 0/0 alcoholGlycerin ext. man250/0 alcohol ext. man75 O/0 glycerin ext. sheep75 0/0 ,, ,,9 (kept 63 hrs.)250/0 alcoholGlycerin ext. ox

Human saliva,. it

Malt diastase.. ..9

Diastaticvalue

42'0

28528733.9

43'8

27-1

38'521'320'6

18-0

6'9

9.4

17-2

5.9

8'8

5-8

Relative timeof reaching

Violet Brownstage stage

25'4 71'525 6726 6923 6620 5919 6421 6320 6711 4412 43 311 4710'8 40'016 53 l16 52-5'7'2 272-2 24

pancreas, qtuantities of ferment were used which gave achromic pointtimes of respectively 41, 8, and 204 minutes, yet the times required toreach the deep violet stage were respectively 27, 2o and 240/0 of theachromic point time, and to reach the brown stage, respectively 73, 72,and 69'5 I/o. Again, in the case of human saliva, with achromic pointtimes of 4i and 28 minuites, the times requiired for the violet stage were

respectively 17 and 14 0/0, and for the brown stage, 55'S and 49 0/0.In the case of most pancreatic glands, two or more different extracts

were examined, and by comparing the values so obtained amongstthemselves, it will be seen that the nature of the liquid used forextraction is practically withouit influence on the rate of progress of thecolour changes. Means of all the values obtained with each gland havetherefore been taken, and these means are given in the last two columnsof the table. We see here that on an average, extracts of dog's and ofpig's pancreas take about 250 /0 of the achromic point time to digest thestarch to the violet stage, and 69 0/,, to digest it to the brown stage.Extracts of ox and of sheep's pancreas, on the other hand, digest thestarch to the violet stage more thali twice as quickly, they taking onlyabout 11 0/0 of the achromic point time. With them the brown stage isalso reached much more rapidly, i.e., in about 42 0/o of the achromic

157

Mean relativetime of reaching

Violet Brownstage stage

25'4 71'5

24'7 67'3

20'0 63'3

11'3 44-7

10'8 40'0

16 52'7

4'6 25'5

1. M. VERNON.

point time. These very striking differences in the rate of progress ofthe colour reactions may perhaps be better realised by taking a concreteinstance. Thus with an achromic point time of 10 mintutes, extractsof the former glands would take 21 minutes to digest the starch to itsviolet stage, whilst those of the latter would take only just over aminute. The former would take nearly 7 mniniutes to digest it to thebrown stage, whilst the latter would take only about 41 minutes.Consequently the further progress to the achromic point would takeonly 3 minutes with the foriner extracts, and nearly 6 minutes with thelatter.

Extracts of human pancreas, whether of man or of woman, showintermediate relationships to those of the two groups of glandsmentioned. The average time of conversion to the violet stage liesnearly midway between them, but that to the brown stage approachesmore nearly to the values of dog and pig's glands. At the bottom ofthe table are given the values obtained with fresh, filtered human saliva,and with malt diastase. The saliva of two different individuals wasused, and though one sample was nearly three times as active as theother, it gave practically the same rate of colour change. The timesrequired to reach the violet and brown stages are distinctly less thanthose for extracts of human pancreas, they lying midway between thesevalues and those obtained with ox and sheep extracts.

Two of a series of values obtained with an aqueous extract of malt(1 part of moalt to 4 of water) are given at the bottom of the table. Thefirst values were obtained when the achromic point time was 10 minutes,and the second, when it was 110 minutes. It will be seen that therelative time re(1uiired to reach the violet stage is much less thau withany of the pancreatic extracts, and also that it is very much reducedwhen the digestion is prolonged. With intermediate times of digestion,intermediate values were obtained, e.g., one of 488% with a digestiontime of 34 minutes. The diastatic value of the extract, calculated andcorrected according to the method described in a former paper', alsodiminished considerably when the digestion time was prolonged. Therelative time required to reach the brown stage was nearly constant,however. In these digestions with diastase it was found very difficultto fix with any certainty the time of disappearance of the colour reactionwith iodine, because of its ensuing so gradually. Thus we see thatwith an achromic point time of 10 minuites, more than 7 minutes were

1 This Journal, xxvii. p. 177.

158


required to take the starch from the pale brown stage to the achromicpoint.

It may be thought that the differences observed in the progress ofthe colour reactions are in some way connected with the differences inthe diastatic value of the extracts used. Thus we see from the secondcolumn of the above table that the extracts of pig's pancreas were themost active, then' that of the dog, then those of human pancreas, thenthose of the sheep, and lastly that of the ox. As a rule, therefore, thesmaller the diastatic valuie, the quicker the relative time of onset of theviolet and brown stages. However, the values obtained with individualglands prove that there is no direct connection between diastatic activityand progress of colour reactions. Thus the alcoholic extracts used wereinvariably much less active than the corresponding glycerin ones, yetthey gave practically the same colour change times.

The rates of progress of the colouir reactions may continue almostunchangred even when the conditions of action of the ferment areconsiderably altered. For instance, it was showti1 that by the additionof *2 0/0 or more of sodium chloride, or of -0)4 0/0 of hydroclhloric acid tothe dilute starch, the efficiency of the pancreatic ferment might be verygreatly increased. In the present series of observations we see from theacconmpanying table that the diastatic value of the extracts of pig'spancreas, acting in 20/0 NaCl, was on an average 4-1 times greater thanthat of the sarne extracts acting in ordinary tap water, whilst that oftihe extracts of sheep's pancreas was 2-9 times greater. Neverthelessthe mean times of digestion to the violet and brown stages do not differin any case by more than 2 6 °/o from the normal tap water values. The

Relative time Mean relativeRelation of reaclhing tiuie of reachingto tap _

Diastatic water Violet Brown Violet BrownExtract value value stage stage stage stage

750/0 glycerin ext. pig+ 2 0/0 NaCl 1269 4-5 26 63,, " (kept 24 hrs.) ,, 1305 4.5 25 638 25-3 64-7

25 0/0 alcohol ext. pig ,, 108 3-2 25 68)75 0/0 glycerin ext. sheep + *2 0/0 NaCl 75.7 3-6 11 44

(kept 63 hrs.) , 51A4 2-9 9*4 43 10.5 45250/0 alcohol ext. sheep ,, 14-2 2-1 11 48750/0 glycerin ext. pig + i004 ole HCI 1218 4.3 32 71 30 5 71-525 0/0 alcohol ,, ,, 78A4 2-3 29 72r750/0 glycerin ext. sheep ,, 55x1 2a6 14 49 1425 0/0 alcohol ,, ,, 11-7 117 14 561 52*5

1 This Journal, p. 184.

159

H. M. VERNON.

addition of -004 0/0 HCl was not, as a rule, nearly so effective as that of*2 0/0 NaCl, and also it produced a slight but distinct effect on the colourchange rates. Thus all of the mean values are froin 2 7 to 7-8 0/0 greaterthan the corresponding tap water values.

Of the other conditions of action investigated, viz., temperature anddilution, the former was found to exert a marked influence on theprogress of the colour reactions. Series of observations were carried olutat 200, and at 500 or 60°, as well as at 38°. From the accompanyingtable we see that at 20° the digesting starch progresses to the violet andbrown stages relatively more slowly than it does at 380, whilst at 600 itprogresses very much more rapidly. With a 1X) minute achromic point

Glycerin extract Alcohol extract

Diastatic Violet Brown Diastatic Violet BrownTemperature value stage stage value stage stage

Pig 200 209 34 72 23-7 33 71380 285 25 67 33-9 23 66600 132 9A4 52 11-2 5 0 35

Sheep 200 14-1 23 57 3-5 23 6238° 20-6 11 44 6-9 11 4750° 14-0 5*7 36 413 5-3 32600 *56 *15 6-5 -

timne, extracts of pig's pancreas acting at 200 took over 3 minutes toconvert the starch to the violet stage, and 7 minutes to the brown stage,whilst at 600 they took from a to 1 minute for the former stage, and 3jto 5 minutes for the latter. The extracts used must have containedsmall quiantities of deleterious substances which especially delayed theadvent of the achromic point at high temperatures, and so hastened therelative time of advent of the violet and brown stages. Thus thealcoholic extract was only about an eighth as active as the glycerinextract used, and so of course about eight times more extract had to beadded to the starch. We accordingly find that at 60° the diastaticvalue of the alcoholic extract was relatively less than that of the glycerinextract as compared with their respective values at 380, whilst the violetand brown stages ensued relatively more quickly. The sheep's pancreasextracts were so much weaker than the pio's, that the deleterioussubstances contained in them almost prevented the starch from everreaching the achromic point, when it was kept at 600. For instance, inan observation with the glycerin extract of sheep's pancreas it was foundthat the violet stage was reached in i minute, the brown stage in 11minutes, and the achromic point in 170 minutes. In an observation

160


with the alcoholic extract, the violet stage was reached in 3 minute, butthe brown stage not for about 200 minutes.

The dilution of the 1 0/0 starch employed to various degrees had verylittle influence on the rate of progress of the colour reactions, but withstarch above 1 0/0 in concentration, both the violet and red-brown stageswere reached with greater rapidity. In the accompanying table jaregiven the results obtained by the action of glycerin extract of pig'spancreas on arrowroot starch (with which all the observations thus fardescribed were made), and also on pure potato starch. It will be seenthat this latter starch gave a somewhat lower diastatic value than the

Arrowroot starch Potato starch

Concentration Diastatic Violet Brown Diastatic Violet Brownof starch value stage stage value stage stage

3 0/<} 197 14 38 176 11 301X5 204 20 51 - - -1.0 201 23 55 - -*5 203 24 63 172 15 50*25 204 25 66 175 13 54*125 188 28 63 171 16 52

former, and also lower percentage times for the violet and brown stages,but in both instances the actual diastatic value obtained was almostuininfluenced by the dilution. This dilution was always made with halfdistilled water, and half tap water, as this was the proportion used inthe other observations.

THE RELATIONS BETWEEN PAVY'S AND FEHLING'S SOLUTIONS.

In order to determine the relationships subsisting between the rateof progress of the colour changes of the digesting starch with iodine,and the rate of formation of maltose, series of estimations of the per-centage of maltose present were made. For this purpose the gravimetricmethod of copper reduction would have been too laborious, and soPavy's method of volumetric analysis', checked by gravimetric analyses,was adopted instead. In this method 10 c.c. of the standard Pavy-Fehling solution are equivalent to 005 grm. dextrose, and not '05 grm.as in the case of Fehling's own solution. Nevertheless the solutioncontains 4'158 grms. of copper sulphate per litre, whilst Fehling'scontains 34'65 grms. In fact, Pavy found that the presence of the

I Pavy's Physiology of the Carbohydrates, p. 73.

161

11PH. XXVIII.

H. AL VERNON.

ammonia altered the reactioni, and that with his solution each moleculeof dextrose reduced six molecules of CuO, whereas with normalFehling's solution it only reduces five. Pavy does not seem to havedetermined the reducing power of maltose by means of his solution, buthe states' that the reducing power of lactose is 60 or a little over, ascompared with that of dextrose taken as 100.

As the result of careful gravimetric analyses, Brown and Heron2came to the conclusion that the reducing power of maltose, as comparedwith that of an equal weight of dextrose taken as 100, was approxi-mately 61. O'Sullivan- found a somewhat higher reducing power formaltose, and concludes that the value is probably 63-16, or one corre-sponding to-a reduction of six molecules of CuO. Still Brow n and Heronfound that by Using the number 61, they obtained the closest possibleagreement between their gravimetric and polarimetric observations, soI have assumed it to be correct. Also Soxhlet4 obtained a similarvalue in some of his experiments. As regards lactose, very variableresults have been obtained. Many observers have found that eachmolecule of lactose corresponds to 7 molecules of CuO, but some, toeven 8 molecules'. Soxhle t fouind that it corresponded to 7A4 mole-cules, and assuming that this is correct, it follows that the reducingpower of lactose, as compared with that of dextrose taken as 100,is 77.9.

With Pavy's solution, the reducing power of lactose in two seriesof observations was found to be respectively 63 71 and 64 8:3 0/0 of that ofdextrose, or on an average, 64-27 0/0. This is a distinctly higher valuiethan that given by Pavy, but possibly he assumed that lactose isanhydrous, whereas, unless specially dried, it contains one moleculeof water of crystallisation. Three series of observations on differentsamples of maltose gave for the reducing power the values 50'24,51-01 and 51'82, or on an average, 51-02%. Comparing Pavy andFehling's solutions, therefore, the reducing powers are:

Pavy FehlingDextrose 100 100Lactose 64 27 (x 3 =77 1) 77.9Maltose 51-02 (x&=61-2) 61

I Pavy's Physiology of Carbohyd., p. 13. 2 Trans. Chent. Soc. 1879, p. 619.3 Watts' Dictionary of Chemistry, Iv. p. 555.4 J. fur praktische Chemie, xxi. p. 227.5 For literature see SoxhIet, Cheem. Centrplbla tt, ix. p. 238. 1878.

162


On multiplying the values obtained witi Pavy's solution for lactoseand maltose by , we get values which approximate very closelyto those obtained with Fehling's solution. As Pavy's solution, inrespect of its dextrose equivalence, contains 1; times more coppersulphate than Fehling's solution, it follows that the two solutionsreact in exactly the same manner with lactose and maltose, or thatin both of them one molecule of lactose reduces 7-4 molecules ofCuO, and one of maltose, nearly 6 molecules.

In all the maltose estimations made by Pavy's method, therefore,the reducing power of the maltose was taken to be x 61 = 50-83 q/oof that of dextrose. This ratio gave results in good agreement withthe few estimations made by the gravimetric method of copper reduc-tion'. For instance in a malt diastase digestion, the volumetric methodshowed that after 70 minutes' action, 73 8 0/0 of the starch was convertedinto maltose, and the gravimetric, also 73-8 0/0. After 6 hours' digestion,the volumetric method gave 79.4 0/0, and the gravimetric, 81-0 0/0.Again, in a digestion with extract of pig's pancreas, the volumetricmethod gave, after 10 minutes' action, 58&2 0/0 maltose, aud thegravimetric nmethod, .597 0/0; after 3 hours', the volumetric gave71 6 o/0, and the gravimetric, 71-4 0/0. In another estimation with pig'spancreas extract, the volumetric method gave, after 31 hours' digestion,72-8 0/0 maltose, and the gravimetric, 7111 0/0. In the particular obser-vations to be described, I believe that the volumetric method gave justas accurate results as the gravimetric would have done. Thus it hasbeen shown by Soxhlet2 that this gravimetric method is liable to veryconsiderable sources of error. Supposing that in glucose estimationsonly just stufficient Fehling's solution is taken, then the copper reduc-tion is some 3 0/ too low; whilst if double as much as is needed beadded, it is about 7 0/o too high. These observations of Soxhlet'swere made with such extreme care and accuracy that there can be nodoubt as to their validity. In the case of a digestion with extract ofpig's pancreas, I found that when 2'3 times the necessary amount ofFehling's solution was used, the copper reduction was 5-3 0/0 greaterthan when only 1-17 times the amount was used.

According to the formula given by Pavy for his solution, eachlitre should contain, in addition to the 4-158 grms. of copper sulphate,20 4 grms. of Rochelle salt, 20-4 grms. of potash, and 300 c.c. of strongammonia. With this solution it requires some skill to complete the

1 See O'Sullivan, Tramn. Chem. Soc. 1876, p. 130. 2 Ibid. p. 220.11-2

1. M. VERNON.

decolorisation of the copper before all the ammonia has boiled off, andRamsden has found' that the method is much more workable if the-amount of ammonia be increased. As used by him, each litre contains,with the same 4-158 grms. of copper sulphate, 40 8 grms. of Rochellesalt, 40 8 grms. of potash, and 600 c.c. of ammonia. Probably such anincrease in the Rochelle salt is unnecessary, and indeed a certainamount of tartrate is precipitated on addition of the ammonia, but Ican confirm him in his conclusion that this solution is a much moreconvenient one than Pavy's own, and I have used it for almost all ofmy observations. In mnaking maltose observations, it is essential forsuccess that the inaltose should be run into the copper solutioncomparatively slowly, so that it is very difficult to get consistent resultswith Pavy's own solution. I have found, in fact, that the reducing powerof maltose with this latter solution is distinctly lower than it is withthe former, it being on an average only about 48,0 0/0 that of dextrose,instead of 50-8 0/0. The reducing power even of dextrose seemed to be1-2 0/0 less, but that of lactose was the same with both solutions.

THE RATE OF FORMATION OF MALTOSE WITH DIFFERENT DIASTASES.

In order to obtain comparable results in the various starch diges-tions, such a quantity of pancreatic extract or of saliva was taken aswould give an achromic point time of exactly 10 minutes. As a rule,200 c.c. of 1 0/0 starch paste, freshly made with distilled water, weremixed with 200 c.c. of tap water (less the volume of extract to beadded), and the whole brought to a temperature slightly above 380.The extract was then poured in, and the flask of liquid violently shakenup. After 1, 2, 3, 5, 10, 30 and 180 minutes' digestion, volumes of50 c.c. of the digest were rapidly withdrawn with a pipette, and runinto small flasks into which 2'5 c.c. of strong ammonia had previouslybeen introduced. This ammonia instantaneously stopped the fermentaction, and its addition also enabled one to work the volumetricanalysis with greater ease. When gravimetric analyses were to be madein addition, the volume of liquid withdrawn was of course larger, andthe ferment in it was destroyed by quickly heating nearly to boiling.Small quantities of the digesting liquid were tested with iodine duringthe first 10 or 12 minutes, in order to make sure that the correctamount of extract had been added.

1 Private communication.

164


Arrowroot starch was employed in almost all the observations, butthe few in which pure potato starch was substituted gave almost thesame results. On drying the starch to constant weight at 1100, twoestimations with the arrowroot starch showed the presence of respec-tively 83 56 and 83-57 0/0 of solid matter, and one estimation with thepotato starch, of 83,35 0/0, whilst incineration of some arrowroot starchshowed the presence of 07 0/0 of ash. Also some 1 0/0 arrowroot starchpaste was inverted by heating for 3 hours with 2 0/0 sulphuric acid,and the glucose formed estimated by Pavy's method. This indicatedthe presence of 8350-0/o pure starch, hence in all the observations tobe described it was assumed that the starch used contained 83 5 0/0 of(C6H10O0),n

Some of the results obtained are reproduced in the accompanyingfigure in the form of curves. Here it will be seen that the rate of

00

so

10~CHO -oa 20 2530_

$CaatOM,pOINt TajE IN Pj 243

Fig. 1.

maltose formation differs considerably with different diastases. It isinteresting to note, however, that at the achromic point time, or after10 minuttes' digestion, the percentage of starch converted into maltosewas nearly the same with the glycerin extracts of sheep, ox and pig'spancreas, and with humxan saliva. Thus it varied only from 56.9 0/0 to

59K7j /a, or almost within the limits of experimental error. To take the

X9a °/0, rams vt h iiso xeietlerr otk h

165

1I. 31. VERNON.

curves separately, it will be seen that only a single curve is given forboth sheep and ox values. This was because they were practicallyidentical. In the series of values obtained with sheep's extract-represented in the curve by crosses-the amount of extract added wastoo small, so that the achromic point was reached in 13 minutes,instead of 10 minutes. Hence the percentage of maltose formedafter 10 minutes, for instance, would have been yielded in 10 x IO

= 7-69 minutes, if the correct amount of extract had been added tothe starch, and so it is entered in the curve at this time value. Thepercentages obtained after 1, 3 and 5 minutes have been similarlyintroduced in the curve at the corrected times. The values givenunder 30 and 180 minutes were in reality obtained after respectively39 and 230 minutes, and so need no further correction. Two otherseries of digestions were carried out with sheep's pancreas extract, andone other with ox pancreas extract, and were found to yield valuescorresponding to those given in the curve. Thus from this curve wesee that the proportions of starch converted into maltose after 5, 10 and180 minutes, are respectively 52-3, 59 7 and 71-3 0/0. The other obser-vations gave 53-3 and 523 0/ conversion after ' minutes; 60-0 and56-4 0/0 after 10 minutes, and 73 0, 69-5 and 72'3 0/0 after 180 minutes.

With glycerin extract of pig's pancreas, the conversion into maltosewas at first much slower than that observed with sheep and ox pancreasextracts, it being, for instalnce, only 25-0 and 44-0 0/0 after respectively2 and 5 minutes, instead of 35 0 and 52 3 0/0. From the achromic pointtime onwards, however, it was practically the same. With humansaliva, the maltose formation was still slower than with pig's pancreasextract for the first ten minutes, but it then shot ahead, and afterthree hours it was found that in three different digestions respectively79 8, 81-3 and 79-1 0/0 of the starch was converted into maltose; i.e., onan average about 10°/o more than with pancreatic extracts. Of thevalues given in the curve, those at 1 and 3 minutes were obtained witha saliva only just a third the activity of that used to obtain the valuesat 2 and 5 minutes, yet the agreement is very good. After 10 and30 minutes' digestion, values of respectively 57 0 and 70-2 0/, wereobtained with the one saliva, and of 56'8 and 71-0 0/o with the other,or within the limits of experimental error, practically the same per-centages. Observations on the rate of conversion of starch into rnaltoseby human saliva have already been made by Chittenden and Ely'.

1 Amer. Chem. Journ. iv. p. 120. 1882.

166


They assumed that the sugar formed had the same reducing poweras dextrose, but correcting for this, their results show that 78-00/ ofthe starch was converted into maltose after 30 minutes' digestion, and86-20/o after 2 hours'. The more rapid hydrolysis observed by themwas doubtless due to the fact that they used 2j to 7 times more salivathan I did.

With vegetable diastase extract, made by extracting ground maltwith twice its weight of water for about 6 hours, and then filtering,the maltose formation was extraordinarily rapid as compared with thateffected by animal diastase. Thus we see that 56 5-0/0 of the starchwas converted in 1 minute, and 74-50/0 in 5 minutes, whilst by theachromic point time the 3 hours' value had nearly been reached. Evenwhen the amount of diastase added is considerably reduced, the maltoseformation is still extraordinarily rapid. Thus in the table given beloware quoted the percentages obtained in a digestion in which theachromic point was reached in 50 minutes. Even then we see thata larger proportion of maltose was fornmed in 3 minutes than in anyof the animal diastase digestions in 10 minutes. In these observationsit was of course necessary to estimate and allow for the maltose presentin the extract itself. The action of malt diastase on starch has alreadybeen worked out with great care by O'Sullivan', by Brown andHeron2, Brown and Morris3, and others, and as far as comparison ispossible, my results are in agreement with theirs. Thus Brown andHeron found that diastase acting on a' /l potato starch at 400, converted79-1 0/0 of the starch into maltose in 30 minutes. The achromic pointtime was not determined exactly, but it was something between 15 and30 minutes. When the temperature of conversion was- 500 or 600 C.,80-4 0/ was converted in 60 minutes.

We have seen that the time relations of the colour reactions of astarch digestion with iodine are affected considerably by temperatureand dilution, but scarcely at all by the presence of sodium chloride,-orthe substitution of alcoholic extracts for glycerin ones. The same istrue as regards the rate of maltose formation. In the accompanyingtable are given the percentages obtained in a digestion with alcoholicextract of pig's pancreas, and also in one with glycerin extract of pig'spancreas acting in the presence of 20°/ NaCl. Though in this lattercase the amount of ferment employed was not a quarter of that needed

I Tranm. Chem. Soc. 1876, p. 125, and 1879, p. 770.' Ibid. 1879, p. 596. 3 Ibid. 1885, p. 527.

167

H. M. VERNON.

in the normal digestion, yet the rate of maltose formation was almostthe same, as can be seen by comparing the figures given in the table.

Malt diastase Glycerin extract of pig's pancreas(60 min. Alcohol ,--

Time of achromic extract of Under normal In '20/0 With *5 O/0 With 30o/odigestion point time) pig's pancreas conditions NaCI At 20' C. potato starch potato starcl1 min. 28'1 11.9 14'3 11.9 16'1 - 34'22 51'8 22'6 25'0 22'3 26'5 23 9 -3 64'1 29'5 31'9 29'2 35'4 - 61'65 70's 40'9 44'0 40'9 47'8 43'110 - 57-1 57-8 54'5 58-9 53 9 67'230 77-4 69'1 67'5 66'1 67'9 64-4 69'3180 81'0 71'9 71'5 71-8 73-2 70'9 74'5

In a digestion effected by glycerin extract of pig's pancreas at 200instead of 380, the figures given in the table show that the rate offormation of maltose was slightly enhanced. At 600C., however, itis very greatly retarded. The dotted line curve in the accompanyingfigure indicates the rate of maltose formation at this temperature withglycerin extract of pig's pancreas, and we see that though at first it wasnearly as rapid as in the digestion at 380, it soon began to fall behind,and after 30 minutes only 59'1 0/0 of the starch had been convertedinto maltose, instea'd of 67'5 0/o. The action of the ferment practicallyceased at this point, for a further 2j hours' digestion only raised theamount of maltose by '3 O/l. In another digestion with glycerin extractof pig's pancreas carried out at about 610 instead of 600, the retardation

rTMjiN mNT#V& rES

Fig. 2.

168


was even greater. Thus after 5, 10 and 30 minutes the proportions ofstarch converted were respectively 33-8, 44-6 and 55.9 0/0, whilst after3 hours the amount was again only *3 0/0 greater than after 30 minutes.

The other curves given in the accompainying figure indicate theeffect of dilution on the rate of maltose formation. All the observationswere made with glycerin extract of pig's pancreas, and arrowroot starcheWe see that in *25 0/0 starch, the rate of formation of mnaltose isvery little slower than in 5 0/o starch. In 1 0/0 starch, however, it isdistinctly more rapid, and in 3 0/0 starch muich more rapid still. Adigestion was also carried out with 3 0/o potato starch, and with it, as,can be deduced from the values given in the above table, the rate ofconversion of the starch into maltose was even quicker than with 30/,arrowroot starch. In fact, for the first 3 minutes it was about as fast asin the slow malt diastase digestion (i.e., that with a 50 minutes' achromicpoint time). With *50/0 potato starch, the rate of maltose formationwas apparently somewhat less than with *5 0/o arrowroot starch, but thedifferences observed are almost within the limits of experimental error.The degree of dilution seemed to have no influence on the percentageof starch converted after 3 hours' digestion, except in the case of 3 0/.starch. In this instance, the average amount was 74'0 /0, or 2-3 0/ogreater than the average amount converted at other dilutions.

If the rates of formation of maltose with different diastases, or withthe same diastase acting under different conditions, be compared withthe relative tinmes of attainment of the colour reactions with iodine, itwill be found that as a rule they run on more or less similar lines. Thusas regards rapidity of maltose formation, malt diastase comes first, thenpancreatic diastase of the sheep and ox, then that of the pig, and lastlysalivary diastase. As regards quickness of onset of the violet and brownstages with iodine, the order is slightly different, it being: malt diastase,pancreatic diastase of the sheep and ox, salivary diastase, and lastlypig's pancreatic diastase. If the observations on pancreatic diastase bealone considered, the relationship is closer. Thus with extracts of pig'spancreas, the proportions of starch converted into maltose after 2j and6j minutes, or when the violet and red-brown stages had been attained,were respectively 29 and 50 0/o; whilst with extracts of sheep and oxpancreas, the proportions converted after Ij and 41 minutes, or at thecorresponding colour stages, were respectively about 24 and 50 0/o.Probably, therefore, under similar conditions of dilution and temperature,each given stage in the colour reactions with iodine has, correspondingto it, a definite amount of maltose formation, whatever the pancreatic

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BH. M. VERNON.

extract employed. When the temperature is changed, the relationshipby no means holds good, for we saw that at 600 the onset of the violetarid brown stages is greatly quickened, whilst the maltose formation isretarded. As regards dilution, we saw that the onset of the violet andbrown stages is quickened in concentrated starch, but this quickeningis not sufficient to correspond with the rapid increase in the maltoseformation. Thus with 30/0 starch, the proportions of starch convertedinto maltose after 1P4 and 3-8 minutes, or at the violet and brown stages,were respectively about 35 and 57 0/0, as against the values of 29 and50 0/0, obtained with *5 O/o starch.

THE ESSENTIAL DIFFERENCES IN THE NATURE OF THE DIASTASES.

The differences observed in the action of the several diastasesexperimented with have now been recorded. It remains for us to,decide how far, if at all, these indicate essential differences between theferments in question. Brown and Heron,' as the result of theirexperiments with aqueous extract of pig's pancreas, came to theconclusion that the course of action was practically the same as withmalt diastase. With this latter ferment they found that there was avery definite stage in the hydrolysis of the starch molecule correspondinato a conversion of 800/o of the starch into maltose, and the few observa-tions I have made confirm this result. They also found a similar stage,torresponding to an 80 0/o conversion, in the digestion of starch bypancreatic extract, whereas in the numerous observations quoted above,we saw that the definite stage in the hydrolysis corresponded to a con-version of slightly more than 70 0/0 of the starch. It is difficult toaccount for this considerable difference of result, but it may perhaps be,due merely to differences in the conditions of action in their and mvexperiments. They refer to their experimental data only in generalterms, without giving details, so I was not able to attempt to imitatetheir conditions exactly. In their malt diastase experiments, theygenerally worked with 5 0/o starch paste, and if this concentration wereused in the pancreatic extract digestions, it may have been the chiefcause of the difference observed. Thus, as noted above, I found thatwith 3 0/o starch there was a distinct increase in the maltose formation,As compared with that obtained with more dilute starch. Unfortunatelyby my method it would have been practically impossible to obtain

1 Proc. RBo. Soc. 1880, p. 393.

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ACTION OF--DIASTASES.

accurate results with starch of greater concentration than this of 30/0, so

I,did not attempt it. Brown and Heron seem to have employedvery much more ferment than I have done, for they say that about 3 c.c.of extract were used for each grain of starch. Even if this be a misprintfor gram, they would still have been using more than ten times thevolume of extract employed by me.

Even more striking than the differences observed in the action ofmalt and of pancreatic diastase at 380, are those occulrring at highertemperatures. Thus Brown and Heron found that malt diastase hadpractically the same action at 600 as it had at 400, it still converting800/0 of the starch into maltose. On the other hand we saw thatpancreatic diastase would only convert 600/0 or less of the starch at thistemperature. At higher temperatures still, malt diastase also has itsactivity curtailed, for Brown and Heron found that at 750 only 300/0 ofthe starch was converted into maltose. By rendering the malt extractslightly alkaline with caustic soda, and allowing it to act at 660, theyreduced its starch converting power to 20 0/, -and so an apparently smallchange in the conditions of action of the ferment may profoundly alterthe course of that action.

It will be noticed that the percentage of starch converted intomaltose in the various experiments was always a multiple of ten, itbeing either 200/0, 300/o, or 80o/0. Brown and Heron accordinglyconcluded that the simplest formula for soluble starch was 10 C12H20O109and that C02112000o groups were successively split off and converted intomaltose. My, own results are obviously in agreement with this hypo-thesis, for I found that human saliva converted 80 0/0 of the starch intomaltose, whilst pancreatic diastase converted about 70 0/0 when actingat 380, and 60 0/o when acting at 600. Brown and Morris' subsequentlyconcluded that the formula of starch was ten times greater than thatabove given, but whatever the formula, there can be no doubt as to thetendency of the starch molecule to undergo hydrolysis in tenths.

The above noted differences in the action of malt and pancreaticdiastases will probably be considered sufficient to indicate that they areessentially different bodies, but there is another argurnent more con-vincing than any of those offered. The one ferment is of vegetableorigin, and the other of animal, and as far as I am aware, no instalice isknown of a complex organic body, such as a proteid, common tomembers of both animal and vegetable kingdoms. In the case

1 Trans. Chem. Soc. 1885, p. 527, and 1889, p. 449.

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H. M.- VERNON.

of the pancreatic diastases themselves, however, one cannot speakwith such certainty. It is true that the digestions effected by themdiffer considerably from each other as regards their reactions with iodine,and their initial rate of maltose formation, but we have seen that if 3 0/,starch be substituted for the usual *5 0/0, the course of the digestion ismore affected than if the pancreatic diastase of the sheep be substitutedfor that of the pig. It is possible, therefore, that the extracts of thegland of each animal contain minute quantities of certain poisonoussubstances, which modify the action of the ferment, and bring aboutthe differences in question. Against this view is the fact that anextract of pancreas which has greatly deteriorated induces just thesame course of action as a powerful extract, though of course itmust contain-proportionally to its activity-very much more of thehypothetical poisonous substances. On the whole, therefore, it seemsreasonable to assume that the diastatic ferment occurring in thepancreas of, for instance, the pig and the dog, is actually a differentbody from that found in the pancreas of the sheep and ox.

The diastatic ferment of human saliva seems to be essentially adifferent body from the ferment of the pancreas, and also from maltdiastase. Thus, like the latter ferment, it converts 800/0 of the starchinto maltose, not 700/0, but on the other hand it has the slowest rate ofmaltose formation during the first few minutes of digestion, instead ofthe fastest.

It may have been noticed that in the above observations it wasalways assumed that maltose was the only sugar formed. In the caseof pancreatic diastase digestions, of course, this is not strictly the case,but Brown and Heron have shown' that the rate of formation ofdextrose is very slow during the first few hours of a digestion, so that onemay for practical purposes ignore its presence. Probably not more thanabout 10 /o was formed in the 3 hours during which the digestions werecontinued, and if the proportions of starch converted into sugar becorrected by this amount, they approach more closely than ever to thetheoretical 70 /0.

SUMMARY.

The progress of the colour reactions of digesting starch with diluteiodine varies considerably according to the nature of the diastaseemployed. With ferment sufficient to give an achromic point time of

I loc. cit.

172


10 minutes, extracts of pig and of dog's pancreas take about 24 minutesto digest starch to the deep violet stage-as tested with iodine-and7 minutes to digest it to the pale red-brown stage. Extracts of sheepand of ox pancreas take only about 1 minutes to digest the starch tothe violet stage, and 41 minutes to the red-brown stage, whilst extractsof human pancreas take intermediate times. Human saliva took about14 and ,54 minutes for the respective stages, whilst malt diastase tookonly about i and 21 minutes.

The rate of change of the colour reactions is not much affected bythe addition of NaCI, or of a minute quantity of HCI, or by varyingthe dilution of the starch from 1 0/0 to *125 0/0, but is greatly influencedby temperature. With glycerin extract of pig's pancreas acting at 600instead of at 380, the times of reaching the violet and brown stageswere respectively 1 and 5 minutes, instead of 24 and 64 minutes, whilstat 200 they were 31 and 74 minutes.

The amount of starch converted into maltose was determined byPavy-Fehling solution, checked by gravimetric copper reduction analyses.With Pavy's solution, the reducing powers of maltose and lactose arerespectively 51-0 and 64-3 0/0 that of dextrose. With this solution,in contradistinction to that of Fehling, each molecule of dextrosecorresponds to six molecules of CuO, not five, but both solutions reactin the same way with maltose and lactose. Thus if the above numbersbe multiplied by 4, they give values of 61-2 and 77-1 0/0 for the reducingpowers of maltose and lactose respectively, i.e., practically the valuesobtained with Fehling's solution.

Taking in all cases sufficient ferment to give an achromic pointtime of 10 minutes, it was found that the rate of conversion of starchinto maltose by extracts of sheep and ox pancreas is more rapid, atfirst, than with extract of pig's pancreas or with human saliva, but atthe achromic point time the amount converted varied only from 59*7to 56 9 0/0 with the different ferments. After 3 hours' digestion at 38°,the amount converted by the pancreatic extracts was always slightlyover 700/0, whilst with human saliva it was 800/0. With malt diastase,the maltose is formed so very much more rapidly that after 10 minutes790/0 of the starch was converted. After 3 hours, however, in con-firmation of the results obtained by Brown and Heron, it was foundthat only slightly over 800/o is converted.

When 10/0 or 30/0 starch is used instead of the usual *5 0/o, theinitial rate of maltose formation is considerably increased, but the

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final amount of starch converted is practically unchanged. Also thesubstitution of potato starch for arrowroot has very little effect.

With extract of pig's pancreas acting at 60° instead of at 380, only600/0 or less of the starch is converted into maltose, instead of 70 /0.

It will be seen that the results are in good agreement with Brownand Heron's hypothesis that the starch molecule undergoes 4ydrolysisin tenths.

It is concluded that malt diastase is an essentially differentbody from animal diastase; that salivary diastase is different frompancreatic, and that probably the pancreatic diastases of variousanimals are different bodies.

Documents

Magdalen College, Oxford. (Two Figures in Text.)