studies on enzyme action. xi.-hydrolysis of raffinose by acids and enzymes

Studies on Enzyme Action. XI.-Hydrolysis of Raffinose by Acids and EnzymesAuthor(s): H. E. Armstrong and W. H. GloverSource: Proceedings of the Royal Society of London. Series B, Containing Papers of aBiological Character, Vol. 80, No. 540 (Jun. 23, 1908), pp. 312-321Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/80277 .

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312

Studies on Enzyme Action. XI. -Hydrolysis of Raffinose by Acids and Enzymes.

By H. E. ARMSTRONG, F.R.S., and W. H. GLOVER, Ph.D.

(Received and read April 2, 1908.)

[International Catalogue of Scientific Literature.

Authors' title slip:-D. Q. Subject slips:-

D 1820 Cane-sugar. Hydrolysis by acids and enzymes. D 1830 Raffinose. Hydrolysis by acids and enzymes. D 8014 Invertase, action of, on raffinose and sucrose compared. D 7095 Cane-sugar and raffinose, hydrolysis of, by acids and enzymes compared. Q 1240 Invertase, action of, on raffinose and sucrose compared.]

In a previous communication of this series (vol. 74, p. 191),* it is pointed out that the various glucosides are hydrolysed by acids at very different

rates, the relative values being approximately of the order shown in the

following table:-

a-Methylglucoside .................. 100 /- , .................. 180

a-Methylgalactoside ............... 540 3- ............... 880

Salicin (a /-glucoside) ............ 600

Milk-sugar (a /-galactoside)...... 720 Maltose (an a-glucoside) ......... 740

Cane-sugar, it is to be remembered, is hydrolysed at a rate vastly more

rapid-at least 1000 times as rapidly as maltose, in fact. These differences,

taking into account the peculiar specific behaviour of enzymes as hydrolytic agents, raise questions of interest from the chemical side and they are of no

slight significance perhaps also from a biological point of view. The two stereo-isomeric methylglucosides are represented by the following

formule:-

H OH H OH C C CC--- I I I I

/ I \\ HR -1/l \\H H CH30.C.H pH.C-C--C.OH./

/ OHH \/ OHH

0a-Methylgl e

a-Methylglucoside r-M ethylglucoside

*All references are to these ' Proceedings.'



Studies on Enzyme Action.

It will be seen that the two radicles H and OCH3 shown as attached to the one carbon atom merely occupy different or reversed positions relatively to the two other radicles with which the carbon atom is connected.

As to the manner in which the hydrolytic attack takes place, two views are possible: the one being that the compound behaves much as the simple ether CH3.0.CH3 would and that the hydrolyst becomes associated with the

oxygen atom to which the CH3 group is attached; the other being that the attachment is to the oxygen atom in the ring. On the former view, it is to be supposed that the two isomeric compounds would be hydrolysed with

equal readiness, as the CH30 groups are equally weighted; but on the latter, it is conceivable that the methoxy-group is less easily accessible to hydrolysis in the one case than it is in the other; or it may be that an inductive

effect is exercised by the one oxygen atom upon the other which renders the

oxygen atom in the ring either more or less attractive of the hydrolyst, as the case may be.

In the galactosides, the CH30 group occupy the same relative positions as in the two glucosides; but the contiguous oxygen atom in the pentaphane ring must be supposed to occupy a position slightly different from that which it

occupies in glucose, its connection with the carbon atom on the right being slightly different, as shown in the two following formulhe:-

H OH H 0 EI I I i I

/C C\ C ( I --^ H H /I-- . H H

/H,O 1111\ pH.C-C-C.OH. CfL C-C--C.OH. CHjO.C.H /3H.--C-:C.OH. CH30.C.H C0 CC.01H. \\ / HOH \ /R/E OHH

\ / \/ /Aa \0/ \0/

a-Methylglucoside a-Methylgalactoside

Apparently, seeing that the galactosides are the more easily hydrolysed, this difference is sufficient to render the hydrolyst-the attack of which in all probability is directed from the oxygen atom in the ring-more accessible to the neighbouring CH30 group than it is in the glucosides. The behaviour of the glucosidic acetates appears to be in accordance with such an interpre- tation.* In models of glucose and galactose constructed with tetrahedra to

represent the carbon atoms, if these atoms are arranged in a vertical plane the

oxygen atom in the ring occupies somewhat different positions more or less outside this plane; the difference in the relative positions of the OCH3 group and the oxygen atom in the ring, according as the position of these on either side of the ring plane is varied, then becomes apparent.

* Cf. Armstrong, E. F., and Arup, 'Chem. Soc. Trans.,' 1904, vol. 85, p. 1043.

2 B 2

313



314 Prof. H. E. Armstrong and Dr. W. H. Glover. [Apr. 2,

There can be little doubt that considerations of the order here pictured are more or less applicable to the explanation of differences such as those under discussion and that it may be possible eventually, proceeding on these lines, to discriminate between alternative formulah applicable to compounds such as

glucose and galactose. It is from this point of view that the study of

hydrolytic changes is of supreme importance. Enzymes probably act much in the same way as acid hydrolysts; the

attachment of the enzyme, however, appears to be more general and

thorough, so to speak, than that of the acid hydrolyst and presumably extends over a large part of the molecule (conpare III and X, B, vol. 79, p. 361). But in both cases the attack is directed, it may be supposed, from the oxygen atom in the pentaphane ring adjoining the group which undergoes hydrolysis.

In the hope of obtaining further information bearing on this refined

problem, the behaviour of raffinose towards acids and enzymes has been studied in comparison with that of cane-sugar, raffinose being a triose formed of cane-sugar weighted by the attachment of a molecule of galactose.

Iaffinose is a reserve material which accompanies cane-sugar in the sugar beet, in cotton seed, in barley and in wheat, for example. It can either be resolved into galactose and cane-sugar by a special enzyme or it can be resolved into fructose and a biose isomeric with cane-sugar-melibiose-by the action of invertase, the enzyme which resolves cane-sugar into glucose: and fructose.

Hydrolysis of Cane-sugar and Raffinose with the aid of Acids.

Raffinose is easily hydrolysed with the aid of acids at ordinary temperatures, the products being fructose and melibiose, the latter, like maltose and

milk-sugar, undergoing change only at higher temperatures. The experiments with acids were carried out at 25? C. in the manner

described by Caldwell (A, vol. 78, p. 285), with an improved apparatus an account of which will be given at an early date by Messrs. Caldwell and

Whymper. Except where stated otherwise, the polariscope readings were taken with the aid of a spectroscopic eyepiece, using a mercury lamp as the luminous source, the light being of the refrangibility of the dominant line in the green. As raffinose is less soluble than cane-sugar, solutions of quarter molecular strength were used. A complete record of two experiments is

given on the left-hand side of Table I. The columns on the right contain the values of the velocity constant deduced by means of the formula

105 a K = 1 logio t a-xc



Table I.-Hydrolysis of Cane-sugar and of Raffinose by Acids at 250 in Solutions containing 1000 grammes of Water

+ 0 25 gramme molecular proportion of Carbohydrate + 1 gramme molecular proportion of Acid.

Cane-sugar + HIC. Rafinose + HC1. Cane-sugar. Raffinose.

Volumes of the solutions in .c.c ..................... 10738 1084 6 1092 1098'6 1108 9 1148 1

Time. aHg . K. . O3. 2HI.HN03. .C1. HNO3. .NO HO4.

nins. o 7-160 - 27 388 - - - - - - -

20 4-720 502 25-503 421 500 - 465 462 551 - 424 388 393 1445 444 25 4 208 499 25-101 418 49 4666 1 465 552 - 418 388 392 - 442 30 3 720 498 24 700 419 498 44 6 461 466 551 - 422 89 391 446 - 35 3 244 499 24 327 418 497 474 459 465 548 - 420 387 392 - 450 40 2-814 498 23-970 418 504 472 463 465 551 552 420 386 391 448 - 45 i 2-380 500 23-630 418 499 470 460 466 550 554 423 387 394 451 447 50 2 000 498 23-280 421 498 472 459 463 548 555 423 385 391 441 442 55 1 628 498 22-700 418 502 468 464 463 546 551 420 387 393 449 447 60 1-274 499 22 146 418 '496 468 464 459 545 556 420 388 397 445 444 65 0-938 499 21-651 417 500 468 463 460 550 555 419 387 394 442 446 70 0-634 498 21-184 419 503 466 463 459 546 553 418 386 395 443 447 75 0-310 502 20 747 421 503 466 462 460 546 550 419 386 395 446 445 80 0-040 501 20 407 418 504 466 466 461 545 545 418 388 395 445 447 85 -0'213 500 20 106 414 500 466 464 459 550 547 422 387 - 447 447 90 -0 492 503 19-763 417 498 466 462 460 549 550 417 386 - 448 446 95 -0 745 505 19-473 418 501 470 464 458 547 420 - - 444 448

100 -0-942 502 19-200 1 420 500 466 463 460 - 545 417 - - 446 448 105 --1140 501 18 980 419 504 467 464 - - 545 416 - - 443 446 110 --1350 503 - - 502 471 462 -- - 549 415 - - 446 446

c '-4 659 1- 16'695 - - - - - - - -_ -

Means ......... 500 - 418 5 500 468 462 5 462 548-5 550 419-5 393 446 446 s~~i _^ 1__j___

PI3 O,z

C3 cpl (Z



Prof. H. E. Armstrong and Dr. W. H. Glover. [Apr. 2,

The values deduced for cane-sugar are in fair agreement with those obtained by other workers in our laboratory. Contrasting the mean values,

they are as follows:- Nitric acid. Chlorhydric acid. Sulphuric acid.

Cane-sugar ......... 464 500 549 Raffinose ........... 390 419 446

The three acids differ in their activity towards both sugars: the cause of this difference will be discussed in a separate communication, dealing with the sucroclastic action of acids generally.

It is clear that raffinose is less easily hydrolysed than cane-sugar, at a rate

nearly one-fifth less than that at which the latter undergoes change. The ratios for each acid are nearly the same in the case of nitric acid and

chlorhydric acid, but sulphuric acid is relatively less active towards raffinose -a result not without interest, the significance of which, however, need not be discussed here.

Hydrolysis of Cane-sugar and affinose by Invertase.

In contrasting their behaviour towards invertase, comparative experiments were carried out simultaneously with the two sugars under similar conditions. A known volume of a very weak solution of invertase at 25? C. was added to a known volume of the sugar solution of definite concentration, also at 25?, contained in a Jena-glass flask; after adding a few drops of toluene, the flask was corked up and placed in an incubator kept at 25?. At stated intervals, 20 c.c. samples were withdrawn from the flask by means of a

pipette and run into small Jena-glass flasks, each containing a single drop of a strong aqueous solution of sodium hydroxide. By this means the action of the enzyme was at once arrested and equilibrium established between the stereo-isomeric forms of the sugars in solution. The final values were obtained by keeping part of the solution at 25? C. during 24 hours, at the end of which time a drop of sodium hydroxide solution was added as before. In order to obtain the initial values, a drop of sodium hydroxide solution was added to a known volume of the enzyme solution, which was then mixed with the sugar solution in the same proportion as in the experiment.

In the preliminary experiment the raffinose used was that supplied by Kahlbaum. The rotatory powers of the solutions were determined in sodium

light. The results obtained are exhibited in Table II, the values given under K

being those deduced with the aid of the formula K =-t o1,10 a-0 t loa-x

316



1908.] Studies on Enzyme Action.

Table II.

317

Cane-sugar. Raffinose (commercial).

34-2 grammes of cane-sugar (1/10 mol. 59'4 grammes of raffinose (1/10 mol.

Tine. C12H22011) + 4 c.c. of strong invertase ClH.3,016.5H20) + 4 c.c. of strong extract per 1000 c.c. invertase extract per 1000 c.c.

a Per cent. aD' Per cent. K. D hydrolysed. K. a hydrolysed.

mins. 0 4 36 00 0- 12-25 0 0 5 3-88 8-3 753 12 15 1 8 157

15 2-87 25-9 868 11-92 5 9 176 25 2-09 39 5 865 11-51 13-4 249 40 0 78 62-4 1062 11 14 20-1 243 60 -0-13 78-2 1102 10-63 29-3 251 95 -0 87 91 1 1106 9-95 41 6 246

140 -1 -02 93 7 859 9 30 53-4 237 200 -1 10 95 1 656 8-57 66-6 238 260 -1-15 96-0 537 7 95 77 -9 252

o --1-38 100 0 - 6-73 100 0 -

On plotting curves to represent the rates at which the two sugars are

changed, it is seen that whilst one-half of the cane-sugar is hydrolysed in

33 minutes, the half of the raffinose is hydrolysed only after the lapse of

Table III.

Cane-sugar. Raffinose (recrystallised).

34-2 grammes of cane-sugar (1/10 mol. 59'4 grammes of raffinose (1/10 mol. T. 12H 22011) + 4 c.c. of strong invertase C18Ii:-2016.5H0) + 4 c.c. of strong Time. extract per 1000 c.c. invertase extract per 1000 c.c.

a-Ig. Per cent. K. a g. Per cent. hydrolysed. hydrolysed.

mins. 0 4'504 0 0 - 13 768 00 -

10 3-332 17-4 829 13 462 4-6 203 20 2-100 35-6 957 13 168 9'0 204 30 1 074 50 '9 1043 12 -918 12 -7 200 40 0 306 62 3 1058 12 -668 16 4 195 60 -0-848 79 4 1143 -

80 -1 -490 88 9 1193 11 -814 29 2 187 120 -1 -810 93 -6 997 11 -044 40 -6 189 160 -1 -982 96 '2 888 10 -398 50 '3 190 200 -2 -114 98 2 868 9 850 58 5 191

j o -2-238 100'0 - 7065 1000 -



318 Prof. H. E. Armstrong and Dr. W. H. Glover. [Apr. 2,

126 minutes; in other words, it takes about 3-8 times as long to convert one-half of the raffinose into melibiose and fructose as it does to change one- half of the cane-sugar into dextrose and fructose.

For the following experiments the raffinose was purified by dissolving it in hot water, filtering the hot solution, adding a large bulk of alcohol to the filtrate and allowing it to stand during several days. The results are

given in Tables III and IV.

Table IV.

Cane-sugar. Raffinose (recrystallised).

68-4 grammes of cane-sugar (1/5 mol. 118-8 gralmmes of raffinose (1/5 mol.

Tinre. Cl2H220) + 4 c.c. of strong inver- C,lH3206.5H2O) + 4 c.c. of strong tase extract in 1000 c.c. invertase extract in 1000 c.c.

a- g. Per cent. I . a"Il Per centt. hydrolysed. bhydrolysed.

mins. 0 9 886 0 0 - 28'462 0 0

10 8'818 7-7 349 28-156 2 4 106 20 7-548 16-9 402 27 796 5-1 117 30 6 298 25 9 434 27 405 8 3 126 40 5 -198 33 -8 449 26 -972 11 7 136 55 - - - 26-354 16-6 144 60 2 -958 50 1 503 - ' - 70 1 '895 57 7 535 25 -743 21 -4 150 80 0 -985 64 3 560 25 -293 25 -0 156 90 0-262 69 6 574 24 -908 28 0 159

100 -0-366 74-1 587 24-517 31-1 162 115 - - - 23 962 35 -5 165 120 -1 -528 82-5 631 - 130 -1 -932 85 -4 643 23 -352 40 -3 172 140 - 2 266 87-8 653 23 -001 43 1 175 150 -2 '562 89 9 666 22 -667 45 7 177 160 -2 793 91 6 674 22-346 48'2 179 175 -- - -- 21-895 51-8 181 180 -3 172 94 4 694 - 190 -3 302 95 3 699 21 -458 55 2 184 200 -3 446 96 '3 719 21 166 57-5 186 220 - 3 -524 96-9 687 20-662 61-5 188

- a -3'950 100 0 - 15 787 100-0 I _.___ _________ ____ _ .._..__.__,_._..,._.._. __.^.._..__..

On plotting curves solution of one-tenth

representing the rates of hydrolysis in the case of the molecular strength, it appears that whilst 50 per cent.

of the cane-sugar is hydrolysed in 29 minutes, 50 per cent. of the raffinose is

hydrolysed only after 158 minutes, the ratio being 1: 54 as compared with the ratio 1: 3-8 in the experiment with the commercial raffinose. Apparently, the recrystallised raffinose is less quickly hydrolysed than the crude mlaterial. In order to see if this were really the case, comparative experiments wer




carried out, side by side, with the two kinds of raffinose and with cane-sugar; moreover, with the object of ascertaining the effect of further purification on the stability of the sugar in presence of the enzyme, the recrystallised raffinose was dissolved in " conductivity" water, the solution was mixed with alcohol and a stream of carbon dioxide passed through it during 30 minutes, this artifice having been found to be of service in purifying galactose; the solution was then filtered and a large bulk of alcohol added to the filtrate. The material obtained by this method is the "Raffinose III" in the

following table. It was again recrystallised from a mixture of alcohol and " conductivity" water; the material thus obtained is marked " Paffinose IV."

Equimolecular proportions of these samples of raffinose having been dissolved, the liquids were boiled to drive off any alcohol present, and were then diluted to the proper volume. Equal volumes of these solutions were measured out and to each was added the same number of cubic centimetres of a dilute invertase solution. The results are recorded in Table V.

Table V.

Amount hydrolysed at 25?.

1 hour. 2 hours. 3 hours. 4 hours.

Per cent. Per cent. Per cent. Per cent. Cane-sugar .................... 96 2 - - - Kahlbaumll's raffinose ......... 42 -7 65 8 80 3 88 -8 Recrystallised raffinose ...... 38 1 61 9 76 -4 82 -8 Raffinose III ................ 38 4 60-8 76 -9 82 -9 Rafinose IV ................. 37 4 59 2 76 -2 84 -5

"""""""""~~~ !

The various samples of raffinose used in this last experiment had the

following specific rotatory powers:- [a]

Hg

Kahlbaum's raffinose ............ 122-5? Ditto recrstallised ............ 122-4 Ilaffinose III ..................... 121-6 Ilaffinose IV ..................... 122-2

The low value obtained in the case of the third sample may be attributed to imperfect drying rather than to impurity. Probably, the greater activity of the invertase in the case of commnercial raffinose was conditioned by the

presence of traces of " acid " imrpurity (cp. X, p. 362). The results afford clear proof that invertase is a far less effective hydrolyst

of the cane-sugar section of raffinose than of cane-sugar itself, being at least

1908.] 319




five times as active towards the latter. This is most clearly exemplified in the following diagram.

,100o . . .

60 ^^- y'^ -

90 / 708 / C/ e

q) / . w

60 X '

imein minut -e.

bs~ ~~~~~. 0

.

0 0 0 Z 60 40

Two explanations of the difference seem possible: either that the weighting

has had the effect of twisting the oxygen centre or centres from which the

hydrolytic attack proceeds somewhat further away from the junction at which the breakdown takes place on hydrolysis; or that the galactose interposes what is commonly known as steric hindrance, more or less blocking the way,

40 '

as it were, to he hydrolytic agent. Unfotunately we have o certain

0 40 80 -2 0 160 zoo Z40O

Twowledge explanat pesent of the strdiere eve of theossible cane-sugar, and the eighting of deriatives such as raistin tose is altogether robleati the

ydroic Cane-sugar is so differen t f urther awaioses with which it is isoric that

the breakdown takes place on hydrolysis; or that the galactose interposes what is commonly knlown as steric hindrance, more or less blocking the way, as it were, to the hydrolytic agent. Unfortunately, we have no certain

knowledge at present of the structure even of the biose cane-sugar, and that of derivatives such as raffinose is altogether problematic.

Cane-sugar is so different froTm other bioses with which it is isoml-eric that its constitution must be peculiar. The most probable structural and functional formula which can be devised at present is that proposed by Emil Fischer, when written in the following manner:-

(HO)HC--CH(OH) (HO)HC---OCH(OH)

CH2(OH)

(HO)H,C-H- C C------ -O-- C CH-CH(O1)--CH2(OH) O O .

-__ 0/

320



Studies on Enzymne Actioln. Studies on Enzymne Actioln.

The two arrows are introduced to indicate the direction from which

hydrolytic attack may be supposed to proceed: a molecule open to attack in

such manner would, doubtless, be far less stable than one in which only a

single contiguous oxygen centre serves to direct the attack. The hydrolysis of melibiose and of raffinose by emulsin lactase will be con-

sidered in a later communication; the hydrolysis of melibiose by acids and by melibiase will also be dealt with separately.

Studies on Enzyme Action. XII.-The Enzymes of Emulsin.

By H. E. ARMSTRONG, F.R.S., E. F. ARMSTRONG, I.Sc., and E. HORTON, B.Sc.



Authors' title slip :-D. Q. Subject slips:-

D 1850 Amygdalin, hydrolysis by emulsin to amnygdonitrileglucoside. D 801.4 Emulsin (almond)-presence in, of three enzymes. Q 1240 Amygdalase, presence of, in almond emulsin. D 8012 Lactose, hydrolysis of, by glucolactase.

D 6300 Hydrogen cyanide (from amygdalin). D 6350 Glucose (from amygdalin)].

Enmulsin has frequently been the subject of discussion in this series of studies: t. thus the rate at which it hydrolyses milk-sugar was considered in No. II (vol. 73, pp. 507, 515) and its action contrasted with that of Kephir- lactase, which was shown to act more rapidly than emulsin. In No. III

(vol. 73, p. 518) it was pointed out that whilst Kephir-lactase is controlled by galactose and scarcely at all by glucose, emulsin is controlled by glucose and

only to a minor extent by galactose. These conclusions were confirmed by experiments with the methyl-glucosides and galactosides (p. 523). In No. V

(vol. 74, p. 188) the question was discussed whether emulsin proper hydrolyses milk-sugar or whether, as Bourquelot and Herissey have contended, emulsin ontntains a small proportion of lactase: against this assump- tion it was argued that the curve was not of the form to be expected if only a small quantity of lactase were present; that whereas Kephir-lactase was controlled by galactose alone, emulsin was most retarded by glucose and only to a slight extent by galactose, also that the curves for emulsin differed in

The two arrows are introduced to indicate the direction from which

hydrolytic attack may be supposed to proceed: a molecule open to attack in

such manner would, doubtless, be far less stable than one in which only a

single contiguous oxygen centre serves to direct the attack. The hydrolysis of melibiose and of raffinose by emulsin lactase will be con-

sidered in a later communication; the hydrolysis of melibiose by acids and by melibiase will also be dealt with separately.

Studies on Enzyme Action. XII.-The Enzymes of Emulsin.

By H. E. ARMSTRONG, F.R.S., E. F. ARMSTRONG, I.Sc., and E. HORTON, B.Sc.



Authors' title slip :-D. Q. Subject slips:-

D 1850 Amygdalin, hydrolysis by emulsin to amnygdonitrileglucoside. D 801.4 Emulsin (almond)-presence in, of three enzymes. Q 1240 Amygdalase, presence of, in almond emulsin. D 8012 Lactose, hydrolysis of, by glucolactase.

D 6300 Hydrogen cyanide (from amygdalin). D 6350 Glucose (from amygdalin)].

Enmulsin has frequently been the subject of discussion in this series of studies: t. thus the rate at which it hydrolyses milk-sugar was considered in No. II (vol. 73, pp. 507, 515) and its action contrasted with that of Kephir- lactase, which was shown to act more rapidly than emulsin. In No. III

(vol. 73, p. 518) it was pointed out that whilst Kephir-lactase is controlled by galactose and scarcely at all by glucose, emulsin is controlled by glucose and

only to a minor extent by galactose. These conclusions were confirmed by experiments with the methyl-glucosides and galactosides (p. 523). In No. V

(vol. 74, p. 188) the question was discussed whether emulsin proper hydrolyses milk-sugar or whether, as Bourquelot and Herissey have contended, emulsin ontntains a small proportion of lactase: against this assump- tion it was argued that the curve was not of the form to be expected if only a small quantity of lactase were present; that whereas Kephir-lactase was controlled by galactose alone, emulsin was most retarded by glucose and only to a slight extent by galactose, also that the curves for emulsin differed in

321 321



studies on enzyme action. xi.-hydrolysis of raffinose by acids and enzymes

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