on the optimum hydrogen ion concentration and temperature of the style enzyme of pecten maximus

On the Optimum Hydrogen Ion Concentration and Temperature of the Style Enzyme ofPecten MaximusAuthor(s): Alastair GrahamSource: Proceedings of the Royal Society of London. Series B, Containing Papers of aBiological Character, Vol. 108, No. 755 (Apr. 2, 1931), pp. 84-95Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/81526 .

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84

612.322. I

On the Optimum Hydrogen Ion Concentration and Temperature of the Style Enzyme of Pecten maximus.

By ALASTAIR GRAHAM, M.A., B.Sc., Department of Zoology, University of

Sheffield.

(Communicated by E. J. Alien, F.R.S.-Received December 9, 1930.)

Within the past decade there has been a considerable increase in the amount of work produced dealing with the processes of digestion in many groups of animals and more especially of invertebrates. Despite the continuous increase in the interest taken in the subject, and in the number of workers engaged in research on this aspect of invertebrate physiology, there has been little corre-

sponding advance in the methods of investigation employed, and few general results have been obtained. Facts concerning enzymes known to biochemists have only too frequently not been appreciated by biologists. The most frequent method of investigating the digestion processes of an animal has been the

preparation of a crude extract of an entire gut or gland by grinding with sand and diluting either with distilled or with sea water, setting up a series of such

preparations-which possess a hydrogen ion concentration of unknown mean-

ing-at temperatures varying from 0? C. to 100? C. for a length of time dictated more by convenience than by significance, and determining, frequently by not the most delicate method, the amount of digestion that has occurred. The

investigation of effects due to the hydrogen ion concentration has been mostly conducted at that temperature which had been previously found to give maximal digestion and for a length of time again devoid of meaning. As a result of this method of procedure, it is often hard to correlate the optimal PE values and temperatures obtained with those that are actually to be encountered in the living animal.

In the digestion of foodstuffs by enzyme action there are in all five variable factors upon which the process depends :-

(1) The strength of the enzyme in the digestion mass.

(2) The concentration of the substrate.

(3) The temperature at which digestion is carried on.

(4) The hydrogen ion concentration of the medium.

(5) The time for which the enzyme acts.



Optimum Hydrogen Ion Concentration of Pecten maximus. 85

The interdependence of several of these factors has been shown by numerous workers. It has long been known that the activity of an enzyme depends on the temperature at which it is working and also on the hydrogen ion concentration of the medium. S0rensen (1909) using the invertase obtained from vertebrate liver showed that the optimum hydrogen ion concentration varied with the length of time for which the enzyme was allowed to act, and O'Sullivan and Tompson (1890) also using invertase proved that it was dependent on the

temperature at which it acted. A reciprocal connection between hydrogen ion concentration and temperature was later discovered by Compton (1915) working with taka-diastase. The relationship between optimum temperature and time has been recently worked out by Berrill (1929) for the amylolytic enzymes of tunicates, demonstrating that the longer the enzyme is allowed to act the lower becomes its optimum temperature. Miss Nicol (1930) has

repeated some of the work of both Compton and Berrill with the diastase of Sabella pavonina, but obtained no definite positive results.

Apart from the work of Berrill and of Miss Nicol just referred to, very little endeavour has been made by zoologists to relate the various factors under which animal enzymes work. By far the greater number of pg and of temperature optima that have been published recently, therefore, are special cases- statements that at a definite p, and for a certain digestion time a certain

temperature is optimal for the activity of the enzyme, and only too frequently the cases selected have little biological significance. The optimum hydrogen ion concentration for an enzyme being dependent on the temperature at which it is working, it is a point of purely academic interest that a certain pu is optimal at a temperature which the animal can never experience. In order that the results given in digestion work may be significant, the experiments must be based on preliminary investigations determining the time for which the food is exposed to the action of the enzyme in the alimentary canal of the animal, the actual hydrogen ion concentration of the gut, and the temperature at which the animal normally lives. The other two variables-concentration of the substrate and of the enzyme-are beyond the control of the animal and are therefore of only secondary interest to biologists.

An attempt has been made, in the series of experiments about to be described, to investigate more fully the conditions that determine the optimum pE and

temperature values, and to correlate these with the actual conditions found in the living animal. For this purpose the enzyme chosen was the diastase contained in the crystalline style of the scallop, Pecten maximus. This enzyme was selected because it was easy to obtain in large quantities, was of



A. Graham.

considerable strength, and was by far the purest invertebrate enzyme obtainable. In addition, it was a diastase the estimation of the activity of which meant the estimation of reducing sugars, for which more dependable methods exist than for the estimation of other digestion products. Pecten maximus was chosen as the source of the style as it could be easily obtained at Plymouth and because the style was a more or less permanent structure. The animals were kept in circulating water and remained healthy; 3 or 4 days were allowed to elapse after the molluscs were brought in by the trawler before any of that batch were used for experimental purposes, as it was found that that time was

required for the elaboration of a new style, the old one being lost on transference from the sea to aquarium conditions.

The styles were removed from the desired number of individuals by cutting into the stomach through the digestive diverticula and picking out with a pair of forceps. They were carefully washed in sea water to remove any adherent tissue of the digestive diverticula, the mid-gut or the gonad, which might have introduced other enzymes. The styles were then dried on a clean piece of butter muslin, weighed, ground up with a small quantity of silver sand that had been freshly washed either with distilled or with sea water, and made into an extract of usually 0 3 to 0 5 per cent. by weight, by addition of either distilled or sea water, according to circumstances.

The temperatures of the experiments were controlled by the use of a thermostat that was found to be constant to about 1? C. over a 6-hour period. Measure- ments of the temperature were taken at the beginning and at the end of each

experiment, the mean of the two values being used when plotting curves. For

temperatures lower than 20? C. use was made of the circulation of sea water at Plymouth which was found to be constant to 0.1? C. over long periods, of an ice chest and of ice and water mixtures.

The hydrogen ion concentration of the medium was controlled at first by making a sea water extract of the styles and adding HC1 or NaOH directly, the PE being measured colorimetrically by Clark and Lubs' indicators. It was found that such systems were not sufficiently buffered and that incubation at

temperatures over 30? C. led to considerable rise in the PH, presumably due to carbon dioxide being driven off by heat. This occurred even after the mixture had been thoroughly equilibrated by bubbling compressed air through it for 30 minutes, and also after letting enzyme and substrate stand for an hour at 30? C. before mixing. Recourse was therefore had to buffer solutions; those of Walpole (Clark, 1924), of Palitzsch (Clark, 1924), of Atkins and Pantin

(1926), and of Northrop as modified by Miss Nicol (1930) were used, the first

86




and the last proving the most useful, borax or borate buffers seemingly inter-

fering with the digestion processes. By these means the p, of the experimental tubes was kept steady throughout the course of the experiments, colorimetric measurements of 2 c.c. lots at the beginning and at the end of the experiments

giving almost identical tints. As substrate 1 per cent. starch solution free from reducing sugar was used,

and the experiments, which were carried out in test-tubes of 15 c.c. capacity closed with clean corks, were sterilised by the liberal addition of toluol. The

sugar resulting from the digestion of the starch was estimated by the modifica- tion of the Hagedorn and Jensen (1923) method devised by Boyland (1928). This is a much more satisfactory method of measuring reducing sugars quanti- tatively than that of Benedict, which has been so extensively used, as the final titration is an iodine-thiosulphate one, giving a sharp end-point and so allowing accurate results to be obtained.

In the experiments the relations between the concentration of the enzyme and of the substrate and the other variables were not investigated, as it is

improbable that they are under the control of the animal. The amount of

food eaten varies quite without reference to any other of the factors, while the secretion of enzyme is also probably variable without significance.

All experiments were rigorously controlled.

(a) Relation between Duration of the Experiment and Optimnum Temperature.

This relationship was not investigated. It has been known since Blackman

published his results in 1905 that the optimum temperature shows a gradual lowering with increase in the duration of the experiment.

(b) Relation between Duration of the Experiment and Optimum p,.

Serensen (1909) described a change in the optimum hydrogen ion concen-

tration for the activity of the invertase from a vertebrate liver. This change occurred between 2 and 3 minutes after the commencement of the experiment; it can therefore be regarded as of only chemical interest, and it does not seem

possible that such a change in optimum pu could have any importance from

the biological point of view. In order to investigate the possibility of change in the optimum Pu with

variation in the time over which the experiment was conducted, the following series of experiments was performed:-



A. Graham.

Controls.-5 c.c. buffer + 5 c.c. 1 per cent. starch + 1 c.c. 0 3 per cent.

style extract + toluol, boiled and left 51 hours at 30? C. Estimation of sugar in 2 c.c. lots.

Experiments.-5 c.c. buffer + 5 c.c. 1 per cent. starch + 1 c.c. 0' 3 per cent.

style extract + toluol, left at 30? C. for 8, 16, 24, 32, and 51 hour periods, boiled. Estimation of sugar in 2 c.c. lots.

These results when plotted so as to show the relationship between PH and the amount of starch digested give fig. 1, and they show very plainly that pH 6 5

o 8 kours. 3- 5- I\ * 16 do.

E- . / \ 0t 2 - do.

I=5~ 24 hours A\3hous, 52 do.

amount of s \ t g f 2rs

60 -

o.0 5 6 7/ g P q

FIG. 1.-Curve illustrating the relation between optimum pH and the duration of the experiment. Each curve represents a time value. 0, 8 hours; *, 16 hours; 2], 24 hours; * , 32 hours; A, 51 hours.

is optimal by a considerable excess over the others for all times from 8 to 51 hours. When plotted so as to bring out the relation between time and the amount of starch digested they give fig. 2. Here, throughout the course of the experiment, the curve for Pa 6 5 is higher than the others. The curve for

pH 5 6 is also throughout higher than that for pa 7 *4, showing that digestion

88




is generally more rapid on the acid side than on the alkaline. As there is no intersection of the curves, there is no change in the optimum pE, with variation in the time of exposure of the substrate to the enzyme.

3'-

30 -

50- /.

21-^5^o - -o 7G

10 -

00-^ - ,-" 8 -

0 . 10 /5 20 25 )0 5 10 . 5 o Hours 5j

FIG. 2.-Curve illustrating the relation between optimum pH and the duration of the

experiment. Each curve represents a PH value. 0, PH 4.6; *, PH 5 6; *,pH 6.5; 0, PH 74; *, PH 84.

(c) Relation between pE and Optimum Temperature. In order to investigate this relation, the following series of experiments was

performed. In it the strength of the enzyme solution and the duration of the experiment are admittedly merely convenient, as they were found to give a reasonable amount of reducing sugar to estimate, using 2 c.c. as an aliquot part, and as the thermostat was very constant over this period.

Controls.-5 c.c. buffer + 3 c.c. 1 per cent. starch + 2 c.c. 0 3 per cent.

style extract + toluol, boiled, left at temperatures as in the following figures for 6 hours. Sugar estimated in 2 c.c. lots.

Experiments.-5 c.c. buffer + 3 c.c. 1 per cent. starch + 2 c.c. 03 per cent.

style extract + toluol, left at temperatures as in the following figures for 6 hours, boiled. Sugar estimated in 2 c.c. lots.

Experiment 1 buffered at pE 7 3, when plotted so as to show the relation between temperature and starch digested, gives fig. 3 with an optimum temperature at 380 C.



A. Graham.

Experiment 2, buffered at PH 6 2, when similarly plotted, gives fig. 4 with an

optimum temperature at 36? C.

0-1-

E L_

,0'3-

o 0-

0.1?

-A -- --- a .- - \

0o 20 .0 ih 7 6 FIG. 3.-Curve illustrating the optimum temperature at pH 7 3.

4o ?C. 80

10

0 20 h 60 ?C.

FIG. 4.-Curve illustrating the optimum temperature at PH 6 2.

Experiment 3, buffered at pu 4*8, gives an optimum temperature at 26? C., fig. 5.

These three experiments were all conducted separately with enzyme preparations from different batches of molluscs. In order to standardise conditions further the following experiment was carried through, using one preparation

_ A i I J , J

. I

90




of enzyme throughout, so that the ratio of enzyme to substrate was identical in each experimental tube.

0.2 -

O./ 02- /

O 00 20 )0 Lo 5O bo 7b ?C.

FIG. 5.-Curve illustra,ting the optimum temperature at PH 4*8.

Controls.-5 c.c. buffer + 3 c.c. starch 1 per cent. + 2*5 c.c. 0 3 per cent.

style extract + toluol, boiled, left at temperatures as in fig. 6 for 6

hours. Sugar estimated in 2 c.c. lots.

Experiments.-5 c.c. buffer + 3 c.c. 1 per cent. starch + 2-5 c.c. 0 3 per cent. style extract + toluol, left at temperatures as in fig. 6 for 6 hours, boiled. Sugar estimated in 2 c.c. lots.

The results of three experiments, buffered at Pu 4 4, pja 5-3 and pu 6 8, when plotted to show the relationship between temperature and amount of

starch digested, give the three curves in fig. 6. Though in two of them, those for Pu 5 3 and Po 6 8, the absolute optimum has obviously been missed, yet there is a clear indication of a gradual rise in the optimum temperature for the activity of the enzyme with a rise in the PH of the medium. The m6re alkaline the medium, the higher the optimum temperature.

The following point must be carefully noted. When all the experimental tubes are buffered at PH 4*4, then, among these, that incubated at about 25? C. will show maximal digestion; similarly, when all are buffered at p, 6 8, then,

among these, that incubated at about 35? C. will show maximal digestion. But that tube, buffered at P 6 8 and incubated at 25? C., though not optimal among its own lot, will show more digestion than even the optimal one from those buffered at Pu 4 4. The 25? C. optimum is only an optimum in relation

to other tubes at the same or similar hydrogen ion concentrations.



A. Graham.

There is therefore a direct relationship between the p, of the experimental medium and the temperature optimum recorded, and, as is emphasised by

/'50-'-^ * . ^^

00 , 0 10 20 30 4O 50 (0 C. Y

Fia. 6.-Curve illustrating the relation between optimum temperature and the PH of the experiment. Each curve represents a PH value. *, PH 44; U , p, 5*3; *A, pH 6 8.

Compton (1915), the hydrogen ion concentration of the medium must be

specified when any figure is given as the optimum temperature for an enzyme.

(d) Relation between Hydrogen Ion Concentration, Temperature and Duration

of the Experiment. In order to correlate the three variables-time, temperature and hydrogen ion

concentration-with the conditions actually found in Pecten, the following type of experiment was performed. Preliminary investigations had shown that the mollusc lived normally at a temperature more or less constantly of 13? C., and that the food in the gut was in probable contact with the style enzyme for a period of about 10 hours. A series of experiments was therefore carried out, in which the enzyme was allowed to act for the length of time over which it acts in the scallop and at a temperature as close to that at which the animal

normally lives as it was possible to obtain. Under such circumstances the PE

optimum was to be determined.

Controls.-5 c.c. buffer + 4 c.c. 1 per cent. starch + 2 c.c. 0-5 per cent.

style extract + toluol, boiled, left at 15 2? C. for 10 hours. Sugar estimated in 2 c.c. lots.




Experiments.-5 c.c. buffer + 4 c.c. 1 per cent. starch + 2 c.c. 0 5 per cent.

style extract + toluol, left at 15.2? C. for 10 hours, boiled. Sugar estimated in 2 c.c. lots.

The results are shown in fig. 7. The optimum hydrogen ion concentration under such conditions is in the neighbourhood of Pa 5.8. Taking the figures given by Yonge (1925)-and verified for this batch of animals-the hydrogen ion con- F centration of the stomach contents of Pecten

2.0 - maximus and of the mid-gut, the two regions of the Lamellibranch gut where the style enzyme is effective,* are respectively p 5-6 and PH 6.0.

Under the correct natural conditions of time and of temperature, the optimum hydrogen ion concentration experimentally lo- found for the style enzyme is discovered to be the actual concentration encountered in those parts of the alimentary canal of the o.-_ animal where that enzyme is normally effective.

In the case of extra-cellular enzymes, the

optimum p, as usually determined is never ; 7 8 S

very remote from the pu of that portion of FG. 7.- Curve illustrat the optimum PH at natural tempera-

the alimentary tract where the enzyme is ture and duration. found-in Pecten, for example, the optimum as determined at a temperature of 35? C. for a period of 6 hours is about 6*8, a little more than one unit of pE from that of the stomach. In the case of intracellular enzymes, however, the optimal hydrogen ion concentration and

temperature are often values at which the animal could certainly not exist in

a healthy state. Thus Yonge (1926) for the protease of the digestive diverticula of the oyster gives optimum PH values of 3 7 and 10*8. Yonge (1924) has also given the optimum temperature for the enzyme of the hepato- pancreas of Nephrops as 53? C. While it is admitted that the conditions

surrounding an intracellular enzyme, once the tissue containing it has been

destroyed as a tissue, present few, if any, points of comparison with those

* Absorption of the products of digestion, as Dr. Yonge has pointed out, is probably confined to the digestive diverticula.



94 Optimum Hydrogen Ion Concentration of Pecten maximus.

in the intact cell, there is a possibility that if one could determine the true time for which the enzyme normally acts on the ingested particles, the

temperature at which the animal normally lives and the actual hydrogen ion concentration of the interior of the cell, then the optimum PH and temperature for the enzyme would be found to correlate with the natural conditions in these cases as in the case of Pecten. It is, in any event, hoped to

investigate the case of such an enzyme more thoroughly.

Summary.

(1) The diastase of the crystalline style of Pecten maximus has been studied to investigate:-

(a) The effect of time on the pu optimum. (b) The effect of pu on the temperature optimum. (c) The effect of time and temperature on the pu optimum.

(2) The principal results are as follow:-

(a) There is no variation in the pH optimum with variation in the time of the experiment.

(b) There is a fall in the optimum temperature accompanying a fall in the

pr of the medium.

(c) When two out of the three factors-time, pH, and temperature under the control of the animal-namely, time and temperature, are made equal to those in natural conditions, and the optimum of the third is deter-

mined, it is found to be the actual condition encountered in the living animal.

The work described in the previous pages was carried out at the Plymouth Laboratory. My thanks are due to the British Association for permission to use their table there; to Dr. E. J. Allen, F.R.S., and to Dr. C. M. Yonge for

help and encouragement while working there, and in particular to Mr. A. D. Hobson for continued suggestions and advice.

REFERENCES.

Atkins, W. R. G., and Pantin, C. F. A. (1926). ' Biochem. J.,' vol. 20. Berrill, N. J. (1929). 'Brit. J. Exp. Zool.,' vol. 6. Blackman, F. F. (1905). 'Ann. Bot.,' vol. 19. Boyland, E. (1928). ' Biochem. J.,' vol. 22. Clark, W. M. (1924). " The Determination of Hydrogen Ions," Baltimore. Compton, A. (1915). 'Proc. Roy. Soc.,' B, vol. 88, pp. 259, 408.



Physiological Action of Cyanide. Physiological Action of Cyanide.

Compton, A. (1921). Proc. Roy. Soc.,' B, vol. 92, p. 1.

Hagedorn, H., and Jensen, H. N. (1923). ' Biochem. Z.,' vol. 137. Nicol, E. A. T. (1930). ' Trans. Roy. Soc.,' Edinburgh, vol. 56. O'Sullivan, C., and Tompson, F. W. (1890). 'J. Chem. Soc.,' vol. 57. Sorensen, S. P. L. (1909). ' C. R. Lab. Carlsberg,' 6dition franqaise, vol. 8.

Yonge, C. M. (1924). 'Brit. J. Exp. Zool.,' vol. 1. -- (1925). 'J. Mar. Biol. Ass.,' vol. 13.

- (1926). 'J. Mar. Biol. Ass.,' vol. 14.

58I. 2.04:58. I92. 2

The Physiological Action of Cyanide.-I. The Effects of Cyanide on the Respiration and Sugar Content of the Potato at 15? C.

By CHABLES S. HANES* and JOHN BARKER, from the Low Temperature Research Station, Cambridge.

(Communicated by Dr. F. F. Blackman, F.R.S.-Received December 15, 1930.)

I. INTRODUCTION.

The interest which attaches to the physiological action of cyanide arises from the strong inhibitory effect upon the respiratory processes of the cell. This inhibition of cell respiration is interpreted as a specific poisoning of the

catalysts responsible for tissue oxidations, since effects of a similar nature are well known in many oxidation systems of both biological and non-biological origin.

Amongst the variety of animal and plant tissues studied, an inhibitory action of cyanide upon respiration has been observed in all but a few cases, and attention has been focussed almost entirely upon this effect. The extent of the inhibition appears to vary considerably according to both the kind and condition of the tissue, and at present the interpretation of these differences remains somewhat controversial. This aspect of the action of cyanide will

not, however, be discussed in the present paper since we shall be concerned more particularly with an effect of cyanide which has not been recognised hitherto, although possible indications of its existence are to be found in the observations of other authors.

Compton, A. (1921). Proc. Roy. Soc.,' B, vol. 92, p. 1.

Hagedorn, H., and Jensen, H. N. (1923). ' Biochem. Z.,' vol. 137. Nicol, E. A. T. (1930). ' Trans. Roy. Soc.,' Edinburgh, vol. 56. O'Sullivan, C., and Tompson, F. W. (1890). 'J. Chem. Soc.,' vol. 57. Sorensen, S. P. L. (1909). ' C. R. Lab. Carlsberg,' 6dition franqaise, vol. 8.

Yonge, C. M. (1924). 'Brit. J. Exp. Zool.,' vol. 1. -- (1925). 'J. Mar. Biol. Ass.,' vol. 13.

- (1926). 'J. Mar. Biol. Ass.,' vol. 14.

58I. 2.04:58. I92. 2

The Physiological Action of Cyanide.-I. The Effects of Cyanide on the Respiration and Sugar Content of the Potato at 15? C.

By CHABLES S. HANES* and JOHN BARKER, from the Low Temperature Research Station, Cambridge.

(Communicated by Dr. F. F. Blackman, F.R.S.-Received December 15, 1930.)

I. INTRODUCTION.

The interest which attaches to the physiological action of cyanide arises from the strong inhibitory effect upon the respiratory processes of the cell. This inhibition of cell respiration is interpreted as a specific poisoning of the

catalysts responsible for tissue oxidations, since effects of a similar nature are well known in many oxidation systems of both biological and non-biological origin.

Amongst the variety of animal and plant tissues studied, an inhibitory action of cyanide upon respiration has been observed in all but a few cases, and attention has been focussed almost entirely upon this effect. The extent of the inhibition appears to vary considerably according to both the kind and condition of the tissue, and at present the interpretation of these differences remains somewhat controversial. This aspect of the action of cyanide will

not, however, be discussed in the present paper since we shall be concerned more particularly with an effect of cyanide which has not been recognised hitherto, although possible indications of its existence are to be found in the observations of other authors.

* Senior Student of the Exhibition of 1851, at the Botany School, Cambridge. * Senior Student of the Exhibition of 1851, at the Botany School, Cambridge.

95 95



on the optimum hydrogen ion concentration and temperature of the style enzyme of pecten maximus

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