Adsorption from Solution by Ash-free Adsorbent Charcoal. VI - The

ADSORPTION FROM SOLUTION BY ASH-FREE ADSORBENT CHARCOAL. VI. ADSORPTION O F INVERTASE' BY ELROY J. MILLER AND SELMA L. BANDEMER

I n a previous paper2 it has been shown that acids adsorbed on activated ash-free charcoal are incapable of inverting either sucrose that is likewise adsorbed on the charcoal or sucrose in solution in contact with the charcoal. It seemed worth whi:e to investigate further into the cause of the complete inactivity of the adsorbed acid and discover, if possible, whether the inability of the adsorbed acid to invert sucrose is due to non-ionization or possibly to unfavorable orientation of either or both the adsorbed acid and adsorbed sugar. An investigation of the question of whether sucrose would be inverted by adsorbed invertase suggested itself as a possibility of obtaining additional information on the subject. A perusal of the literature revealed the fact that whereas no studies had been reported on the behavior of adsorbed acids and sugar on charcoal, a number of investigators had studied the effects of adsorption of enzymes by charcoal on their activity. Like the reports on adsorption of electrolytes from solution by charcoal, those on the adsorption of invertase were found to contain many anomalous results and contradictory conclusions. Michaelis and Ehrenreicha studied the effect of the type of adsorbent on the nature and extent of adsorption of a number of enzymes using kaolin, talc, blood charcoal, bone charcoal, aluminum hydroxide, and ferric hydroxide. They found that invertase was strongly adsorbed from neutral, alkaline, and acid solutions by blood charcoal and bone charcoal. The invertase so adsorbed was still able to invert sugar. That the inversion was produced by the adsorbed invertase was shown by the fact that the filtrate from the charcoal was inactive. Eriksson4 and Hedin,5 on the other hand, found that serum, egg albumin, and charcoal inhibited the action of invertase. Eriksson showed that the inhibitory effect of animal charcoal on the activity of invertase depended somewhat on the order in which the charcoal, invertase, and sugar were added. The greatest effects were obtained when the charcoal and invertase were first mixed and allowed to stand for some time before the addition of sugar. They attributed the loss of activity t o the combination of enzyme with adsorbent. Beard and Cramer6 showed that glass beads have a similar inhibitory effect on the inverting power of invertase. They explained this on the basis Published by permission of the Director of the Experiment Station ae Journal Article No. 41 n.s. E. J. Miller and S. L. Bandemer: J. Am. Chem. Soc., 49, 1686 (1927). a L. Michaelis and M . Ehrenreich: Biochem. Z., 10, 283 (1908). A. Erikason: Z. physiol. Chem., 72, 313 (1911). S. G. Hedin: Z. physiol. Chem., 60, 82 (1909); 82, 175 (1912). 8 E. Beard and W. Cramer: Proc. Royal Soc., 88B, 575 (1915).

ADSORPTION BY ASH-FREE CHARCOAL

266;

of concentration changes brought about by the large surface of the glass beads. Armstrong and Armstrong,’ however, suggested that the effect was due to substances dissolved from the glass beads. Griffin and Kelson8 repeated the work of Beard and Cramer with glass beads and observed that the hydrogen-ion concentration of the invertase solution decreased when the glass beads were added. They, therefore, attributed the inhibitory effect to the dissolving of alkaline substances from the glass beads and the consequent change in reaction from the optimum acid one to an alkaline one in which invertase is inactive. Griffin and Kelson then repeated the work of Eriksson with animal charcoal and invertase and concluded that there, too, the results were explainable on the basis of changes in the hydrogen-ion concentration of the solution produced by the charcoal. They also repeated the work of Michaelis and Ehrenreich with relatively large quantities of charcoal and confirmed their findings that the invertase was all removed from solution but still retained its activity despite its adsorption by the charcoal. When small enough quantities of charcoal were used so that the adsorption of sugar was negligible the activity of the invertase was unaffected by the charcoal. Selson and Griffin9 extended the work of Griffin and Xelson and presented additional evidence in support of their conclusion that the inhibitory effect was a matter of change in hydrogen-ion Concentration and that the activity of invertase was not affected by adsorption on solids such as charcoal or on colloids such as serum, egg albumin, saponin, and aluminum hydroxide. MichaelislO reported additional data confirming his earlier work and that of iUeyerhofll and Selson and Griffin. That adsorption of invertase by ferric hydroxide and charcoal had no inhibiting effect on its activity thus seemed to be firmly established and Michaelis offered the folloiving conclusion: “One of the conditions associated with colloidality is that the disperse phase contains molecules in the surface which are active and others in the interior which are inactive. The active mass is, therefore, not proportional to the concentration of the substance but to its surface. This condition is not present in an invertase solution. In invertase the active mass of the ferment is, whether ‘free’ or adsorbed on ferric hydroxide or charcoal, always proportional to its concentration. This property justifies the application of the law of mass action.” Simultaneously with the publication of this article by Michaelis, ?;elson and Hitchcock’* reported that they were unable to reproduce the findings of Selson and Griffin with invertase on charcoal and aluminum hydroxide. Although they kept the hydrogen-ion concentration of their solutions constant, the activity of the invertase was now found to be very much less when adsorbed. In the case of the earlier results obtained by Griffin and Nelson with charcoal, the possibility that the invertase had not been completely adsorbed E. F. Armstrong and H. E. -4rmstrong: Nature, 95, 4 2 j (191j). E. G. Griffin and J. M . Selson: J. Am. Chem. Soc., 38, 722 (1916). 9 J. M. Xelson and E. G. Griffin: J. Ani. Chem. Soc., 38, 1109 (1916). ‘OL. Michaelis: Biochem. Z., 115, 269 (1921). l1 0. Meyerhof: Arch. ges. Physiol., 157, 2 j I (1914). J. M. Nelson and D. I . Hitchcock: J. Am. Chem. Soc., 43, 19j6 (1921) 7

2668

ELROI' J. MILLER AND SELMA L. BASDELVER

offered an explanation for the absence of retardation. Selson and Hitchcock were, however, unable t o account for the non-retardation observed by Griffin and Nelson when the invertase was completely adsorbed by aluminum hydroxide. In their experiments retardation was always observed under such conditions, although the retardation was considerably diminished when the aluminum hydroxide-invertase complex was not allowed t o settle to the bottom of the Hask, thereby eliminating the necessity for the diffusion of the sugar to the aluminum hydroxide-invertase complex in the bottom of the flask as a factor in the retardation. They offered as a tentative explanation the suggestion that the rate of diffusion of sucrose to and invert sugar from the enzyme combined with the adsorbent and the rate of hydrolysis of sucrose by the invertase on the adsorbent were factors determining whether or not adsorption retarded the activity. They felt that no evidence had so far been obtained that indicated that the true activity of the invertase, as distinguished from the resultant rate of hydrolysis of the sucrose in the mixture, was influenced by the combination of the invertase with the adsorbent. When it is recalled that most of these studies had as their motive (implied a t least) the elucidation of the nat'ure of enzyme action, it is not surprising that concordant results were not forthcoming and it is still an open question whether the hydrolysis of sucrose by invertase should be considered as proceeding in a homogeneous or heterogeneous medium. The invertase was, of course, the unknown and indefinable quantit,y but in the light of subsequent knowledge so also were the adsorbents. It' is well known from recent studies that hydrous aluminum and ferric oxides vary greatly in their properties, depending upon their mode of preparation, and often investigators were not able to reproduce one another's results in the preparation and use of these adsorbents. The charcoal used in the enzyme adsorption studies was even less well defined than the hydrous aluminum and ferric oxides. In none of the studies was the purity of the material or its activity taken into account. The unknown and variable activity, ash, adsorbed acids, and previous heat treatment were undoubtedly important factors contribut'ing to the conflicting results reported. That such is the case is clearly indicated by the data presented in this paper, The information gained in recent studies on adsorption from solution by charcoal seemed to afford a means of explaining the discrepancies and possibly of securing additional information on the subject. Accordingly, an investigation of the problem x a s undertaken using the ash-free adsorbent charcoals described in previous papers which are bett'er defined than the ones formerly available and used by previous investigators. I t is the purpose of this paper to present the results of this study. Materials and Methods The charcoals anti other materials used in this investigation were, for the most part, the same as those previously used and described in the paper2 on the non-inversion of sucrose by adsorbed acids. Any exceptions d l be noted in the appropriate place. The ash-free charcoals were always ignited at a tem-

ADSORPTIOK BY ASH-FREE CHARCOAL

2669

perature of 1000' or higher just prior to use and their activity and freedom from adsorbed impurities were tested from time t'o time by the usual methods.13 Unless otherwise stated purified blood charcoal having an adsorpt'ion capacity s acid from I O O cc. of solution by 0.25 g. of charcoal was of 5 2 cc. 0.02benzoic used in this n-ork. Invert sugar was determined by the S~haffer-Hartmannl~ modification of the Benedict method. An attempt t o use a polarimeter indicated, as did that of Kelson and Hitchcock,'* that consistent results could not be obtained by its use with invertase-sugar solutions. Tests were made to determine whet'her invertase solutions would reduce the Schaffer-Hartmann copper solution and thus interfere with the determination of invert sugar by t,his method. They mere found not t o reduce the copper solution. This is in keeping with the findings of Hudsonlj that invertase does not reduce Fehling's solution. The invertase preparat,ions were from two sources. Several different lots of a commercial solution used for sugar analysis were used with and without further purification. The remainder of the invertase preparations were prepared from pressed yeast by autolysis using chloroform and the lead acetatcpot,assium oxalate treatment'j for purification. Distilled water was used in the dialysis which vias carried out in collodion sacks for four to six days. The collodion sack was suspended in as small a vessel as possible and distilled water was run in at the top and siphoned off from the bottom at a rate that held the level of the water near the top of the vessel. The sack was thus continuously bathed with practically pure water over most of its surface. The denser solution formed on dialysis could be seen to settle rapidly t o the bottom without a great deal of diffusion. Toluene was used as the preservative during dialysis and storage. The stock solutions thus obtained were further treated before use in a manner which will be described later. They contained 0.41 70 or less of solids which on burning left a residue of approximately 176 ash. One cc. of one of the stock solutions (freshly prepared) which accordingly contained 0.0041 gram of invertase was sufficient to invert five grams of sucrose completely in one hour at 3 ; " . Others were not as active a s this. It has been shown by Sorensen16 and confirmed by others" that the optimal hydrogen-ion concentration for invertase action is between pH 4 anti pH j. In all the experiments herein reported the reaction of the digestion mixture of charcoal, sucrose, and invertase was carefully maintained at pH 4.0 to pH 4 . 2 by means of sodium citrate-hydrochloric acid buffer mixtures. Since it had been found in aninvestigation of adsorption from buffer solutionsi8 that ash-free charcoal changes the p H values of these buffers toward the E. ,J. Miller: J. Phys. Chem., 30, I 162 (1926). I'P. A . Schaffer and -4.F. Hartmann: J. Biol. Chem., 45, 375 (1920-1921). l b C. S. Hudson: U. S. D. A. Circ. 50, Feb., page 2 (1910). S. P. L. Sorensen: Biochem. Z., 21, 131 (1909). I'L. Michaelis and H . Davidsohn: Biochem. Z.,35, 386 (1911); H. A. Fales and J. 51. Xelson: J. 4 m . Chem. Soc., 37, 2769 (191s); R. Willstatter, J. Graser and R. Kuhn: Z. physiol. Chem., 123, I (1922). E. J. Miller and S. L. Bandemer: J. P h p . Chem.,'32, 829 (1928). l3

2670

ELROY J. MILLER .4ND SELMA L. B A S D E X E R

alkaline side, it was necessary to use a mixture sufficiently acid so that after addition of the charcoal the desired reaction would exist in the digestion mixture. Under the conditions obtaining in this work, a buffer mixture of pH 3 . j was found t o be suitable. All the digestions were carried out in j 70 sucrose solutions because it has been shown by Kelson and VosburghIg that a t this concentration the velocity of inversion is greatest. We should, therefore, be able to detect the presence of very small amounts of invertase under these conditions. I n trials with as little as 0.01 cc. of stock solution the easily measurable quantity of 4 milligrams of invert sugar was found and even with o . o o j cc. two milligrams were found after digestion. With later invertase preparations which contained less yeast gum and yet were more active, 0.01cc. produced as much as 3 2 milligrams of invert sugar. In all the work the digestions w r e carried out in Pyrex Erlenmeyer flasks in a water bath at 37' for a period of one hour. While these conditions of hydrogen-ion concentration, concentration of substrate, and temperature may not be precisely the optimum for a digestion period of one hour they were sufficiently favorable so that, as stated above, the presence of sufficiently small quantities of invertase could easily be detected. Furthermore, these conditions were essentially comparable with those maintained by previous investigators in this field. Procedure At the beginning of this investigation it was planned to determine whether, as in the case of adsorption of acids by charcoal, small amounts of invertase would be completely adsorbed and when digested with sucrose would not produce any inversion and whether when sufficiently large amounts of invertase were used so that the charcoal could not adsorb all the invertase from solution, inversion would be produced only by the invertase remaining in solution but not by that adsorbed on the charcoal. I t may be stated at the outset that the parallelism is not complete and that the adsorption of invertase by charcoal differs materially from the adsorption of acids, insofar as the ability of the adsorbed material to invert sucrose is concerned. Under certain conditions all of the enzyme is removed from solution and neither the adsorbed enzyme nor the filtrate from the charcoal has any inverting power. Under others the adsorbed invertase mny retain more than half of its activity. The general procedure for determining whether the adsorbed invertase was still active was briefly as follows: The invertase solution was added to one gram of charcoal, the mixture shaken and evacuated t o removed adsorbed air and insure thorough wetting of the charcoal, and allowed t o stand for one hour with an occasional shaking t o keep the charcoal in suspension. If the volume of invertase solution was less thsn 20 cc., enough water was first added to the charcoal so that the total volume of solution was 20 cc. The charcoal was then filtered with suction on a disc of hardened filter paper in a Gooch crucible. When the charcoal was dry enough to crumble when stirred J. M. Xelson and W C Vosburgh: J. Am. Chem. SOC., 39, 790 (1917)


2671

with a spatula it was transferred together with the filter paper to IOO cc. of solution containing j grams of sucrose and z j cc. of the sodium citrate-hydrochloric acid buffer of pH 3.5. The mixture was digested for one hour at 3 7 " ) again shaking occasionally t o keep the charcoal in suspension. Toward the end of the digestion period the charcoal was allowed to settle and at the end of the period the supernatant solution containing some charcoal was decanted off and cooled for one minute under the tap. It was then filtered thru a Gooch crucible and a n aliquot immediately pipetted into jo cc. of the SchafferHartmann copper solution and the invert sugar determined by the regular procedure prescribed for that method. A blank determination was carried out in exactly the same manner with the single exception that 2 0 cc. of conductivity water was substituted for the invertase solution. The titration value so obtained was subtracted from that obtained in the digestion of the charcoal carrying the adsorbed invertase. To determine whether the invertase had all been removed from solution, one half of the filtrate from the charcoal was made up to a volume of 5 0 cc. containing 2 . 5 grams of sucrose and z cc. of buffer and digested for one hour a t 37' and the invert sugar determined. The value thus obtained was corrected by deducting the value obtained by digesting 50 cc. of solution containing 2 . 5 grams of sugar and 2 cc. of buffer. The amount of invert sugar produced by the same volume of invertase as that added t o the charcoal was determined by digesting it in IOO cc. of properly buffered solution containing j grams of sugar. Results with Unpurified Invertase Experiments were first carried out using a readily available commercial preparation of invertase commonly used for analytical purposes. I t was a clear straw-colored solution. Large amounts of this invertase were adsorbed leaving colorless solutions which when filtered and digested were entirely devoid of any power of inverting sucrose. The adsorbed invertase! however, seemed to possess more or less activity depending upon the amounts adsorbed. The relatively small amounts were almost completely inactivated yet this slight activity persisted even when quantities of charcoal were used in large excess over that necessary to remove all the invertase from solution. In some cases the invertase-charcoal mixtures were digested without filtering, but always there were found these small amounts of invert sugar which, as far as could be determined, could not be attributed to errors in technique. When somewhat larger amounts of invertase 17ere added to the charcoal there was a marked increase in the activity of the adsorbed invertase, but still little or no invertase remained unadsorbed in the filtrates. Wh?n the amounts of inrertase added to the charcoal were further increased the charcoal digestion produced increased amounts of invert sugar, but considerable invertase remained in solution at this point. The data in Table I are illustrative of the results obtained. They confirmed, in part only, the results of those investigators who had reported that invertase retained its activity even

2672

ELROY J. MILLER A N D SELMA L . BANDEhfER

TABLEI Adsorption of Invertase (Cntreated Commercial Invertase) Quantity of Invertase solution added to I gm. charcoal

Amount of invert Amount of invert sugar found after sugar found after digestion of charcoal digestion of carrying adsorbed filtrate from invertase charcoal

Amount oi invert sugar produced by equal amount of invertase in ahsence of charcoal Grams

Grams

Grams

I

0.ooj

0.000

2

0.010

0.000

1.215

5

0.116

0.000

2

IO

0.366

0.002

4.544

15

0.858

0.006

4.918

20

I

285

o 308

25

I

468

I

cc.

607

0.603

5

,882

240

complete inversion

when adsorbed on charcoal. Our results showed that the adsorbed invertase had lost from at least 7 5 % to practically all of its activity. Since very little Kas known about the preparation and purity of this invertase it was deemed desirable to work with a purer product or a t least see whether further purification vould have any effect on the behavior of the invertase, and especially whether the invertase could be completely inactivated by adsorption as was the case with the adsorbed acids A quantity of the original invertase solution was, therefore, dialyzed in the manner above described. Another lot was treated with lead acetate and potassium oxalate in the usual way and then dialyzed. After dialysis the solutions were colorless and clear but a brown flocculent material had settled out and this was filtered off. The solutions were then made up in concentrations as nearly as possible equal to those used in the work with the unpurified material, that is, so equal volumes would produce approximately the same quantity of invert sugar when digested for one hour under the same conditions. Results with Purified Invertase

The results in Table I1 show that the adsorption behavior of the dialyzed and of the puiified and dialyzed invertase x a s decidedly different from that of the untreated material. I n the first place, the adsorbability had been greatly decreased. One gram of charcoal had adsorbed practically all the invertase from 1 5 cc. of the unpurified and left comparatively little in 2 0 cc. whereas from the purified solution 5 cc. was completely adsorbed but a large amount remained unadsorbed in I O cc. In the second place, the invertase adsorbed on the charcoal mas far less active than the unpurified. At the most only 85 milligrams of invert sugar was produced by the charcoal digestion as compared with 1.5 grams or more with the unpurified. Several separate lots of the commercial material were purified and all except one exhibited the same behavior after purification. Usually there were still obtained a few

ADSORPTIOS BY ASH-FREE CHARCOAL

2673

TABLE I1 Adsorption of Invertase (Commercia1 Invertase) (Dialyzed and Purified) Quantity of Invertase solution added to I gni charcoal

cc.

Amount of invert sugar found after digestion of charcoal carrying adsorbed invertase

Amount of invert sugar found after digestion of filtrate from charcoal Grams

Grams

Amount 01 invert sugar produced by equal amount of invertase in absence of charcoal Grams

Dialyzed 0.607

I

0.005

0.000

2

0.005

0.000

I

5

0.005

0.000

2.915

I ,065 2,975 4.575

IO

0.021

15

0,044

20

0.066 0.085

25

5.070

,386

4.515

complete inversion complete inversion complete inversion

Purified and Dialyzed I

0.000

0,000

0.47'

2

0,002

0,000

0.872

0.000

2.258

0.912

3.800

20

0.003 0.008 0.016 0,034

25

0,048

4.279

I

IO 15

2 .

I02

-

2.832

milligranis of invert sugar from the charcoal digestion even when the charcoal was in large excess over that necessary for the complete adsorption of the invertase. Effect of Technique on Results The one exception in which the behavior of a purified and dialyzed invertase solution did not agree with all the others led to the discovery of the reason for the slight activity persistently retained by the small amounts of adsorbed invertase The data in Table I11 show that the invertase in this particular solution, like that in the other dialyzed ones, was much less adsorbed by the charcoal than that in the unpurified solution, but it was decidedly more active when adsorbed on the charcoal than the purified ones. On examination of the solution it was found to be slightly opalescent instead of clear. This immediately strengthened a suspicion that some of the invertase might be associated with large particles of extraneous matter which could be mechanically carried down by the charcoal without being actually held on the charcoal by adsorp tion forces. The particles causing the opalescence were unquestionably larger than the invertase particles in the clear solutions and it was concluded that if they could be filtered out without removing all the invertase, the

ELROY J. MILLER AND SELMA L. BASDEYER

2674

TABLE I11 Adsorption of Opalescent Invertase before Treatment with Charcoal Quantity of Invertase solution added to I gm. charcoal


Amount of invert sugar found after digestion of filtrate from charcoal

Amount of invert sugar produced by equal amount of invertase in absence of charcoal Grams

CC.

Grams

Grams

I

0.009

o 004

0.

2

0

I ,2 7 5

2 0

0.018 o.oj8 0.118 0.166 0.306

25

0.5'7

5 IO 15

00;

o 009 2

13.5

4 062 4 385 4 9'5

j62

2.615 4.50

4.68 5.17 -

After treatment with charcoal to remove opalescence I

0 000

0.000

0,551

13.5

0.130

-

I

0.000

2 ' 505 0.000

5

0.000

0.003

20

0

-

invertase remaining would show the same general adsorption behavior as the other purified solutions. This proved to be the case. Portions of the opalescent solution were treated with small amounts of charcoal and filtered. The solutions were no longer opalescent and when these clear solutions were used in the adsorption experiments the invertase was all removed and, as the data at the end of Table I11 show, the adsorption of the invertase from the one and five cc. samples resulted in complete removal of the invertase from solution and the adsorbed invertase was entirely inactive The invertase in the 2 0 cc. sample was not all adsorbed, however, and the adsorbed invertase possessed considerable activity Unfortunately, there was not a sufficient quantity of the clarified solution to carry out the complete series of tests t o compare with those of the opalescent material Thereafter, however, either the stock invertase solution or the diluted solution was shaken with a small amount of charcoal and filtered through a mat of charcoal in a Gooch crucible just before the adsorption experiments were to be carried out. This served the additional purpose of completely removing the preservative (toluene) from the solution, thus precluding the possibility of the readily adsorbed toluene interfering with or cutting down the adsorption of the invertase. One other precaution was adopted somewhat later after consideration of the possible factors that might be responsible for the activity of the adsorbed invertase. That was the possibility that the addition of a concentrated or stock solution of invertase directly to the charcoal might result in an uneven distribution or piling up of invertase on portions of the charcoal which con-

2675

-4DSORPTION BY ASH-FREE CHARCOAL

TABLE IV Effect of Dilution of Invertase Solution on its Complete Adsorption by Charcoal Quantity and concentration of invertase added to charcoal I 2

3 5 30 40 50 60 50

., f’ ’)

’’ dilute

”

,> ))

,,

0.735 1,351

2 . I49 4.086 3 ’ 993

ceivably might result in a variation in properties of the adsorbed invertase. Some evidence for this was found and is presented in Table IV. The stock solutions were, therefore, diluted just before use so that larger volumes of solution could be added to the charcoal, and also, the charcoal was first wetted with a little water to facilitate a more uniform contact of all the invertase with the charcoal. TVhen this technique was employed it was found that large amounts of purified and dialyzed invertase were adsorbed and the adsorbed invertase had completely lost its power of inverting sucrose. The data in Table V were obtained with an invertase solution that had been prepared from pressed yeast by the method stated above. Similar results have been obtained with every invertase solution that has thus far been tested.

TABLEV Complete Inactivation of Invertase adsorbed on Charcoal Quantity of invertase solution added to I gm. charcoal

cc.

Amount of invert sugar found after digestion of charcoal carrying adsorbed invertase Grams



25

0.000

0.000

1.219

30

0,000

0.000

35

0.000

0.000

40

0.000

0,000

501 862 2.058

45

0.000

0,000

2.244

50

0.000

0.000

2.374

60

0.000

0.000

2.943

I . I.

2676

ELROY J. MILLER AND SELMA L. B A S D E M E R

TABLE VI Complete Inactivation of Adsorbed Invertase on Various Purified Charcoals (All Charcoals Ash-free and Freshly Ignited at 1000’) pH of Invertase filtrate

Charcoal

Blood 8.4 Norite 7.4 Darco 7.5 Sugar Merck’s Medicinal 7 . o Merck’s Blood 7.8 Merck’s Decolorizing 8 . 6

Invert sugar found after digestion of charcoal carrying adsorbed invertase Grams

Invert sugar found after digestion of filtrate from charcoal

Invert sugar produced by equal amount of invertase in absence of charcoal

Grams

Gramm

0.000

0.000

2.437

0.000

0,000

2.437

0.000

0,000

0,000

0.000

0.000

0.000

0.000

0.000

2,437 0.5j2 2.437 3.437

0.000

0.000

2.4.37

That the behavior is not characteristic of purified blood charcoal alone, but holds for all types of ash-free charcoals is shown by the data in Table VI. All the ash-free charcoals freshly ignited at 1 0 0 0 ~ or above yielded the same result, namely, the complete inactivation of a considerable amount of adsorbed invertase. As can be seen from Table VII, however, such nas not the case for unpurified charcoals. With the unpurified charcoals there was more or less activity retained by the adsorbed enzyme. The reasons for this will be more conveniently considered in detail later. TABLE VI1 Adsorption of Invertase by Impure Charcoals Charcoal

pH of Invert- Invert sugar ase filtrate found after from charcoal digestion of charcoal carrying adsorbed invertase Grams

Merck’s Decolorizing ( 2 . 4 j y c ash) Merck’s Decolorizing ( 2 . 32y0 ash) Merck’s Blood ( 8 . 2 9 % ash) Merck’s Medicinal ( 2 4 . 5 7 % ash) Darco (29.9757 ash) Norite ( 6 , goyc ash) Merck’s Blood (heated)

Invert sugar found after digestion of filtrate from charcoal Grams

6.4

0.023

6,z

0 .oI

6.8

0.005

0.003

4.1

0.116

0.015

3.8 9.2 6.4

0.069 0,oo;

o.ooo

0.009

0 . 000

5

0.002

Invert sugar produced by equal amount. of invertace in absencr of charroal Grams

2.344

0.0oi

0.000

,,


2677

Causes of Differences in Adsorption Behavior of Invertase With the differences in the adsorption behavior of the unpurified and purified invertase solutions established, the next step was to find out if possible the reaSons for these differences: why more invertase was adsorbed from the unpurified invertase solution than from the purified, and why when adsorbed it retained more of its activity. Since simple dialysis of the original commercial invertase was sufficient 'to produce the changes in properties and the treatment with lead acetate and potassium oxalate followed by dialysis did not change the behavior any further, it was surmised that the change in properties might be due to removal of electrolytes. The reaction of the solutions before and after dialysis was tested and it was found that the original solution contained a considerable amount of acid, presumably acetic, and the dialyzed solutions, of course, had lost this acid. The solutions after dialysis were neutral in reaction. I t was immediately suspected that the presence of the acid might be responsible for the differences in behavior. If the acid T Y ~ Sthe sole factor, then it should follow that acidification of the dialyzed solution would be sufficient to restore the properties possessed before dialysis. An invertase solution had been prepared from pressed yeast, purified, dialyzed, and its adsorption behavior determined. The data for this preparation are given in Table VIII, and from them it can be seen that the adsorption behavior was similar to that of the purified commercial solution (Table 11). The concentration of the acid in the commercial preparation was determined and a portion of the new invertase was acidified with acetic acid to the same concentration. The adsorption data for this acidified solution appear in Table IX. It will be observed that they are the reverse of those obtained with the neutral solution. Much larger amounts of invertase were completely adsorbed and more of the activity was retained by the

TABLE VI11 Adsorption of Freshly Prepared Invertase (Stock solution twice filtered through charcoal filter t o remove extraneous matter) Quantity of inrertase solution added to I gm. charcoal cc


Grams


I

0,000

0,000

2

0.004 0.015

0.000


0.000

0.735 1,351 1.9oj

0 2 0

0.000

3.010

0.258

20

0.090 0.116 0.156

2 ,5

0.210

4.555 4.878 4,925 5 263

3 5 IO

'5

0

2.780

4.438 4.968

'

2678

ELROY J. MILLER AXD SELMA

L.

BASDEMER

TABLE IX Adsorption of Freshly Prepared Invertase acidified with Acetic Acid (Acidity of invertase solution adjusted t o equal that of commercial solution used in obtaining results in Table I, 26.13 cc. o.ozK acetic acid in I O O (:c. solution) Quantity of invertase Amount of invert solution added to I sugar found after gm. charcoal digestion of charcoal carrying adsorbed invertase cc . Grams



0.696

I

0.022

0.000

2

0.061 0.345 0,771 0.839 I , 083

0.000

1.21;

0.000

3,071 4.528 4.983 j . 141

5 IO

15 20

0.000 0.002

0.003

invertase adsorbed from the acidified solution. How completely the invertase was adsorbed from the acidified solutions can be seen from the figures for the digestion of the filtrate. Only two or three milligrams of invert sugar was produced from even the largest amounts as compared with more than four grams with the neutral solution under the same conditions. The figures for the invert sugar produced by digestion of the charcoal are not as striking as those for the filtrate but they show a substantial increase over those for the invertase adsorbed from neutral solution. It seems obvious, then, that the presence of acid was responsible for the difference in behavior of the original commercial solution and the solutions obtained after purification and dialysis. Effect of Adsorbed Acid on Adsorption of Invertase The marked effect of the presence of acid in the invertase solution of preserving the activity of the invertase when it was adsorbed on the charcoal raised the perplexing question of what the mechanism of the protective action might be. Here were the significant facts that the adsorbed acids alone did not have the slightest power to invert sucrose, either adsorbed or in solution, and appreciable amounts of invertase likewise could be adsorbed with subsequent complete loss of inverting power, yet the simultaneous adsorption of both resulted in the preservation of considerable activity of the invertase. I n view of the fact that virtually nothing is known concerning the nature of the structure of invertase, it may seem futile to speculate on the effect of the acid on the invertase, yet the suggestion has been advanced that since invertase is colloidal in nature and exhibits its optimum activity in acid solution the function of the acid is to increase the surface of the invertase. It seems plausible enough that acid might increase the dispersion of the invertase and thus in some way facilitate the adsorption by charcoal, yet it is not so easily seen why the activity of the invertase should be retained. Further doubt

2679

ADSORPTIOX BY ASH-FREE CHARCOAL

was cast upon the dispersion theory when it was found that much the same effect was obtained if the acids were first adsorbed on the charcoal and then the invertase from neutral solution, Under these conditions the possibility of dispersion of the invertase by acid was precluded, yet much of the activity of the invertase was retained after adsorption. The acid adsorbed on the charcoal increased the amount of invertase that could be removed from solution apparently nearly to the same extent that the presence of acid in the invertase did. That this was not caused by displacement of acid from the charcoal which would then be free to act on invertase is certain from the fact that the effect was noticed both with small amounts of irreversibly adsorbed acids and with somewhat larger amounts which were partly displaced. I n Table 9 are data illustrating the results obtained when invertase was adsorbed by charcoal carrying varying amounts of adsorbed acetic, benzoic, and hydrochloric acids. These results are illustrative of a large number obtained in extensive work on this point. Other experiments were carried out in which the amount of invertase was varied while the adsorbed acid was

(20

TABLE X Adsorption of Invertase by Charcoal carrying Adsorbed Acids cc. invertase solution added t o charcoal carrying varying amounts of adsorbed acids)

Amount and kind of acid on charcoal

Amount of invert sugar found after digestion of charcoal carrying adsorbed acid and invertase Grams

Equivalent to cc. o.ozN

5 , I 5 cc. Acetic 10.30 ” 14.86 ” 18.94 ” 22.14 ”

0.536 0.000

”

467 2.06j

”

2.112

0.000

”

0.000

0.000

I ,

50.01 ”

”

99.10

”

I.205

26.79

’’

10.03 ” 20.05

’I

”

120.05 ”

137.78 5.09 I O .2 0

” ” ”

14.95 ” 17.34 ” 20.08 ”

20.33

”

Grams

I . 016

”

2.260 1.832 1,779 0.3jo 0.475 1.054

25.07 ”


’’ ’’ Benzoic ”

1.095 0.929 Hydrochloric 0.979 ” ”

1,

>, 7, 7,

,,

0.000

0,000 0,000

0.241 0.229 0.000

0.000

0.000

1.327

0,233 0.000

2,000

0.000

1.395 1.363

0.000

I ,320

0.018

0.010


2680

ELROY J. MILLER A S D SELMA L. BANDEMER

held constant. In others the quantity of acid was varied in invertase solutions of constant concentration. Other acids also were used. Since the results were in keeping with the ones here reported, they are omitted for the sake of brevity. It will be observed in the results with acetic acid, for example, that j cc. of adsorbed acid on the charcoal was not quite sufficient to cause the complete adsorption of invertase from solution, although the invertase adsorbed retained considerable activity. With I O cc. of acid on the charcoal all the invertase was removed from solution and its activity on the charcoal was increased. Still larger amounts of acid increased the activity on the charcoal, but this effect increased to a maximum and then decreased again. Similarly for benzoic and hydrochloric acids. It is also apparent that there is some variation in the effectiveness of different acids, acetic being appreciably more effective than benzoic. As can be seen from Table VIII, charcoal free from adsorbed acid could not adsorb completely this quantity of invertase and the adsorbed invertase was far less active. Attempts at Displacement and Activation of Adsorbed Invertase When it had been established that both the presence of adsorbed acid on charcoal and the presence of acid in the invertase solution before adsorption had the effect of preserving, to a considerable extent, the activity of the adsorbed invertase, it became of interest to determine whether the activity of adsorbed invertase could be restored by the subsequent adsorption of acid. Experiments to answer the question were carried out under two sets of conditions. Under one, varying amounts of invertase solution were added to the charcoal and after one hour filtered off and tested for any invertase that might not have been adsorbed. Then the charcoal carrying the adsorbed invertase was added to I j cc. of 0 . 0 2 N acetic acid and after one hour filtered and the acid remaining in solution titrated to determine the amount that had been adsorbed. The charcoal carrying adsorbed acid and invertase was then digested with sugar and buffer as usual. The quantity of invertase used was

(IS

cc.

0.02

TABLE XI Xon-Activation of Adsorbed Invertase by Acid N acetic acid added to charcoal carrying varying amounts of adsorbed invertase)

Cc. invertase adsorbed on charcoal

Cc. 0.02 N acetic acid adsorbed by charcoal carrying adsorbed invertase

Grams invert sugar produced by charcoal carrying adsorbed invertase and acid

25

14.3

0.000

30 35 40 45 50

14.3

0.000

00

14.2

0.000

14.25

0 IO00

14.25

0.000

14.20 14.20

0.000 0.000

Grams invert sugar produced by equal amount of invertase in absence of charcoal

0,417

-

2.317 0.000

2681


TABLEX I 1 Kon-Activation of Adsorbed Invertase by Acid (Varying amounts of acetic acid added t o charcoal carrying adsorbed invertase) Cc. invertase solution adsorbed by charcoal

20

,* ,, ,

Cc. 0.02 N acetic arid adsorbed by charcoal carrying adsorbed invertase

Grams invert sugar p!oduced by digestion of charcoal carrying adsorbed invertase and acid

I O .j

0.000

11.3

0.000

12.0

0.000

12.6

0.000

Grams invert sugar produced by equal amount of invertase in absence of charcoal 0.417

,, ,, 21

varied from approximately the largest that could be completely adsorbed by one gram of charcoal to half that amount. The data in Table X I show that these quantities of invertase were so small that they had little or no effect on the adsorption of the acetic acid, the same amount being adsorbed in both the presence and absence of adsorbed invertase. The results show that there was not the slightest activation of the invertase on the charcoal and no displacement of the invertase from the charcoal. Gnder the other set of conditions, the quantity of invertase adsorbed on the charcoal was maintained constant and varying amounts of acetic acid were added Here again (Table XII) there was no activation or displacement of adsorbed invertase by the acid. Since there are in the literature contradictory statements regarding the displacement of enzymes from adsorbents this question was given some attention. A number of substances which for various reasons might be expected to offer possibilities of displacing adsorbed invertase from ash-free charcoal were tried. The charcoal after standing in contact with the invertase solution was filtered with suction and whi'estill damp was placed insolutions of benzoic acid, methylene blue, tannic acid, stearic acid in alcohol and toluene, dextrin, urease, I % Na HPOd in I "/c glycerine, and 0.5 KH-PO?in 0. j 76 glycerine. With the exception of the stearic acid solutions, none of these displaced the slightmt trace of invertase from the charcoal, and in the case of the stearic acid solutions the amount was so small as to be of doubtful significance. I t has been statedS that sucrose displaced adsorbed invertase to some extent but this has also been denied 9. In the present investigation this point was frequently checked on charcoal carrying active, adsorbed invertase which, when digested with buffer and sucrose, produced large amounts of invert sugar After this digestion a portion of the buffer-sugar filtrate was returned to the water bath and after further digestion tested for additional inversion. There was none in any case. The charcoal was then washed with water and with sugar solution and redigested with buffer-sucrose mixture and it was found that the inversion was practically the same as before, showing that the invertase had not been displaced during the first digestion nor by the

2682

ELROY J. MILLER AND SELMA L. BANDEMER

washing afterward. These results, therefore, confirm the conclusion of Xelson and Griffin that sucrose does not displace adsorbed invertase from charcoal. Effect of Length of Time of Contact of Invertase with Charcoal It has been noted by previous workers3 in this field that the activity of adsorbed invertase depended somewhat on the length of time the invertase had been in contact with the charcoal. It seemed worth while to check these results using the ash and acid-free charcoals and also to determine whether the presence of acid in the invertase solution would delay the inactivation I n preliminary experiments it was found that certain quantities of invertase that were incompletely inactivated when adsorbed on the charcoal for one hour, were completely inactivated after eighteen t o twenty-four hours. Tests a t intervals between these two periods showed a gradual drcrease in activity of the adsorbed invertase. K h e n the invertase was adsorbed from invertase solutions containing acetic acid the adsorbed invertase showed, for both the one hour period and the 24 hour period, considerably more activity than when adsorption was from neutral solution, but nevertheless the activity was very low after 2 4 hours, The data which appear in Table XI11 for one and tmentyfour hour periods for neutral and acidified invertase solutions were obtained with a freshly prepared invertase solution. They are not as conclusive as those obtained in the preliminary work on other solutions but since acid was not used in the earlier work these are given because the invertase used with and without acid was from the same stock solution.

TABLE XI11 Effect of Length of Time of Contact of Invertase with Charcoal Quantity of Invertase solution added to I gm. charcoal

cc. 0.02 N acetic acid adsorbed along with invertase

cc.

Amount of invert sugar found after digestion 3f charcoal carrying adsorbed invertase Grams


Amount of invert sugar produced by equal amount of invertase in absence of charcoal

Grams

Charcoal in contact with invertase solution for

Grams I

hour

50

0.00

0.000

0.000

75

0.00

0.020

0.006

3.993 4.646

50

12.70

0.170

0.000

4. I53

75

16.73

0,237

0.000

4.808

Charcoal in contact with invertase solution for 2 4 hours 50

0.00

0.000

0.000

4,

75

0,003

50

0.00 12.22

0,000 0.000

75

17.03

4.525 4.086 4.538

0,039 0.097

0.000

IO1

2683


Effect of Age of Invertase Preparation on Its Adsorption by Charcoal An invertase solution which was prepared a t the time this investigation was begun and with which some of the data in Tables V to XI11 were obtained was tested from time to time over a period of three years to determine whether the results obtained in those experiments could be reproduced. It was found that the results were essentially reproducible for approximately two and a half years. The only noticeable difference was a gradual decrease in activity so that it was necessary to use somewhat larger quantities of the stock solution to secure the desired results. At the end of this period the activity seemed to decrease more rapidly and a change in adsorption properties became noticeable. At the end of three years a fresh preparation of invertase was made for comparison with the three-year old one. I t was prepared in the same way that the old one had been and had approximately the same activity that the old preparation originally had and the yeast gum content was also the same, 0.40%. TABLE XIV Adsorption of Three-Year Old and Freshly Prepared Invertase showing Change of Adsorption Behavior of Three-Year Old Material Three-Year Old Invertase ( 5 0 cc. st'ock solution diluted to j o o cc. for use)

Cc. invertase solution added to I gram charcoal



25

0.000

1.021

50 75

0.000

3 ' 408 4.347

IO0

0.016

0.000


2.424 3 996 4.623 '

complete inversion complete inversion

Freshly Prepared Invertase (4 cc. stock solution diluted t,o

500

for use)

25

0.000

0.000

50

0.000

0.000

75

0.020

0.ooj

2.606 3.993 4.646

IO0

0,035

0.091

complete inversion

Solutions of the old and of the new preparation were made from the stock solution in concentrations such that 25 cc. would invert approximately half of the five grams of sucrose in one hour. It will be noted from Table XIV that it was necessary to use only 4 cc. of the new stock solution for 500 cc. while 50 cc. of the old were required. It will also be noted that the old invertase was completely inactive when adsorbed on the charcoal and only when the largest amount (100cc.) was used was there the slightest. trace of activity exhibited by the adsorbed invertase. The amounts of invert sugar produced by the invertase filtrate from the charcoal showed that while appreciable quantities of the invertase had been adsorbed the greater part remained unadsorbed and active.

2684

ELROY J. MILLER A S D SELY.4 L. BANDEMER

On the other hand, the new preparation behaved in the same manner that all the freshly prepared ones had. Large quantities were completely adsorbed and completely inactivated] and even when the largest amount (100cc.) was added practically all of it was adsorbed and only a small amount remained active on the charcoal. When the old and the new preparations were adsorbed from acetic acid solution there was likewise an appreciable difference in their behavior. The data in Table XV show that the presence of acetic acid in the new invertase, as in previous cases, resulted in the complete removal of the invertase from solution by the charcoal] and the adsorbed invertase retained considerable activity. The old invertase was not nearly so completely removed from solution under the same conditions but there was an appreciable retention of activity by the adsorbed invertase as compared with complete inactivation when adsorbed from neutral solution.

TABLE ST.' Adsorption of Three-Year Old and Freshly Prepared Invertase from iicetic Acid Solution showing Change of Adsorption Behavior of Three-Year Old Material Three-Year Old Invertase ( j o cc. stock solution diluted to j o o cc for use and containing 27.88 cc. S / j o acetic acid per 100cc.) Cc. invertme solution added to I gram charcoal

Amount of acid on charcoal equivalent t o cc. N/ j o



6.97 13.54

0,095

75

17.71

0.220

0.293 0.380 0.631

IO0

21.24

0.626

2.115

25

50

0.000


2.574 4.077 4.807

complete inversion

Freshly Prepared Invertase (4 cc stock solution diluted t o j o o cc. for use and containing 27.40 cc. S / j o acid per I O O cc.) 25

6.85

0,094

0.000

50

12.70

0.I 7 0

0.000

75

16.73 19.92

0.237

0.000

o 986

0.000

IO0

2 641 4.153 4.808

complete inversion

Three-Year Old Invertase ( z o o cc. stock solution diluted to 500 cc. for use and containing 2 7 . 2 j cc. X / j o acid per I O O cc.) 5

0.006

IO

0.024

15

0.056

20

0.081

o.oj9 1.437 3 ' 549 4.616

2,409 3 ' 902

4.737 5 . '93

2685


From these data it seemed likely that the difference in behavior between the old and the new preparations might be due to the relatively large amount of yeast gum in the solutions made from the old stock solution, for when a solution four times as concentrated with respect to the invertase and yeast gum but of the same acetic acid concentration was used, the invertase again was not so completely removed from solution by the charcoal and retained much less of its activity. The data showing these results appear at the bottom of Table XV. It seems quite likely in the light of other results that if the concentration of acetic acid had been increased proportionately to the yeast gum the behavior of the concentrated solution would have been more nearly the same as that of the dilute. Since evidence bearing on the effect of t h e yeast gum was obtained from another angle, extensive work was not done on the effect of the proportion of acid to yeast gum. The results of the few experiments that were carried out, however, supported this conclusion and the data in Table X which show that the nature and quantity of adsorbed acid on the charcoal influences the amount of invertase adsorbed and its activity after adsorption on the charcoal, also seem to indicate the same probability. Role of Yeast Gum in Adsorption Behavior of Invertase Additional evidence that the change in adsorption behavior of the old invertase preparation was due to the large proportion of yeast gum to active invertase was obtained in this manner: A very active preparation of invertase, low in yeast gum, was prepared, and to a portion of this solution sufficient gum arabic was added to make the total solids content approximately the same as in the three-year old preparation. As can be seen from the data in Table XVI the bulk of the invertase was not adsorbed from neutral solution

TABLE XVI Adsorption of Highly Active Invertase in Presence of Gum Arabic (Low yeast gum content. Dialyzed 1 2 0 hours) Cc. invertase Amount of solution added acid on to I gram charcoal charcoal equivalent to cc. 0.025




Neutral invertase solution containing 0.014 g. gum arabic per solution 15

0.00

0.000

20

0.00

25 30

0.00

0.022 0,049 0.061

0.00

3.676 4.334 4.997 5.027

IOO

4.609 5.056

complete inversion complete inversion

Invertase solution containing 0.14 g. gum arabic and 26.83 cc. o acetic acid per I O O cc. solution 15

4.02

20

5.37

25 30

6,71 8.05

0.123 I79 0.191 0.384 0.

2 947 3 508 3.822 4.346 '

cc.

4.298 4.867 4.975 j ,004

02

N

2686


in the presence of gum arabic and the activity of the adsorbed invertase was relatively slight. The fact that activity of this invertase solution was considerably higher makes these results the more significant. I n the same table are shown the results when a solution of nearly the same concentration of invertase and gum arabic was acidified with acetic acid. The magnitude of the effect of the acid was, as with the three-year old invertase, much smaller than in the absence of the gum arabic although the effect of the acid was unmistakably in the same direction, namely, an increased adsorption of the invertase and greater retention of activity by the adsorbed invertase. How great the inhibiting action of the gum arabic on the adsorption in both neutral and acid solutions was, can readily be seen by referring to the adsorption data in Tables XVIII and X I X for this same invertase. The same quantity of acetic acid was sufficient to cause the complete adsorption of these quantities of invertase in the absence of gum arabic. It seems quite clear then that the addition of the gum arabic to the new invertase caused it to behave in the same manner as the three-year old, and the gradual change in adsorption behavior of the-invertase with age was due to the larger quantities of yeast gum involved in the experiments since it was necessary to use larger volumes of the stock solutions to produce the required amount of invert sugar. TABLE XVII Non-Retardation of Invertase Activity by Gum Arabic Cc. invertase solution added to buffer-sucrose solution

'5 20

25

30

Grams invert sugar formed on digestion of invertase solution in absence of gum arabic

4.516 4.853 5.023 5,053

Grams invert sugar formed on digestion of invertase solution containing 0.14 g. gum arabic per IOO cc.

4.484 4.867 j ,007

5.068

It should be noted that the presence of gum arabic in the amounts used in these experiments did not influence the activity of the invertase itself. The data in Table XVII show that the same amount of invert sugar was produced by the invertase in the presence of gum arabic as in its absence. It is exceedingly interesting that gumarabic and yeast gum, twosubstances of presumably different origin and nature, should produce the same effect on the adsorption behavior of invertase. The possible significance of this fact for theories of the structure of invertase will be referred to again in the discussion, but it may be stated here that it seems fairly certain that the gum arabic functions as a protective colloid for the invertase particle. Adsorption of Highly Active Invertase Since the invertase solutions thus far used were of approximately the same activity, total solids content, and adsorption behavior, and had all been prepared by the same method, that of Hudson, it was nenessary to prepare


2687

a product by another method in order to determine whether the method of preparation would influence the adsorption behavior of the invertase. A solution of invertase was, therefore, prepared by the method of Willstatter and Schneider.*O By this method the yeast was rapidly autolysed by using a large amount of chloroform, and the development of a high acidity was prevented by the addition of suitable amounts of ammonium hydroxide during the early stages of the autolysis. After autolysis the solid matter was filtered off and the filtrate was treated as before with lead acetate, and the lead removed with potassium oxalate. Portions of the resultant solution were ,dialyzed for lengths of time varying from 1 2 0 hours to 260 hours. I n carrying out the dialysis it was necessary to keep both the distilled water and the invertase solution saturated with toluene, otherwise changes (presumably bacterial in nature) took place which rendered the invertase solution opalescent. Provision was, therefore, made to agitate the solutions and maintain the necessary saturation. The pressed yeast used in this preparation differed from that used in the others in that it was starch-free. The invertase solutions prepared by this method contained less total solids and yet were more active than the previous ones. They contained 0 . 2 5 % total solids compared with 0.41% in the previous, and 0.01cc. of the stock solution under the usual conditions produced 3 2 milligrams of invert sugar in one hour, as compared with as little as 4 milligrams by some of the earlier preparations. The adsorption data for two solutions of invertase, one of which had been dialyzed for I Z O hours, and the other for 260 hours, appear in Tables XVIII and X I X . The results, on the whole, vary but slightly from those obtained with the solutions prepared by the previous method. Large amounts of invertase were adsorbed from neutral solution and completely inactivated. The presence of adsorbed acetic acid on the charcoal and of acid in the invertase solution again resulted in the adsorption of much larger quantities of invertase and a tendency to preserve the activity of the invertase after adsorption. The difference, if any, in the adsorption behavior of this preparation was that the presence of acid either on the charcoal or in the solution caused the complete adsorption of somewhat greater quantities of invertase, and the preservative effect was somewhat less. This was apparently due to the fact that the yeast gum content of this preparation was only slightly more than half that of the other preparations. The data in Tables XVI and XVII showing the effect of the addition of gum arabic were obtained with this invertase, and it will be observed that the gum arabic reduced very markedly the ability of the acid to increase the adsorption of the invertase. These results may, therefore, be regarded as further evidence that the colloidal properties of the invertase are dependent upon the yeast gum which very likely functions as a protective colloid. They indicate, too, that the more nearly the invertase is freed from yeast gum and concomitant substances the less stable it will be, the greater will be its adsorbability, and the less activity it ?"

R. Willstatter and K. Schneider: 2. physiol. Chem., 142, 257 (192j).

2688


TABLEXS’III Adsorption of Highly Active Invertase (Low yeast gum content. Dialyzed 1 2 0 hours) Cc. invertase solution added to one gram of charcoal

Amount of acetic acid on charcoal equivalent to cc. 0.02 N

Grams of invert sugar found after digestion of charcoal carrying adsorbed invertase Grams

Grams of invert sugar found after digestion of liltrate from charcoal Grams

Grams of invert sugar produced by equal amount of invertase in absence of charcoal Grams

Charcoal acid free and invertase solution neutral 20

0.00

0.010

0.211

30

0.00

0.019

3.010

4.263 4.960

Acetic acid adsorbed on charcoal before adsorotion of invertase 15

4.00

20

5.30 6.6j 7.95

25

30

0.032 0.089 0.176 0,330

0,000 0.000

0.048

Invertase solution acidified with acetic acid, I5 20 25

30

4.07 5.42 6.78 8.14

0.083 0.176 0.342 0.417

3.912 4.263 4.583 4.960

0.000

27.12

cc.

0.02

0.000

0.000 0.000

1; per I O O cc. 3,880 4.263 4.487 j.1 5 0

0,000

TABLE XIX Adsorption of Highly Active Invertase (Low yeast gum content. Dialyzed 260 hours) Cc. invertase solution added to I gram charcoal

Amount of acid on charcoal equivalent to cc. 0.02 N


Amount of invert sugar found after digestion of liltrate from charcoal Grams


Charcoal acid free and invertase solution neutral 15

0.00

0.000

0.051

20

0.00

0.000

I .

063

4.806 5.155

25

0.00

0.042

I.

780

complete inversion

30

0.00

0 . O j I

3,905

),

Invertase solution acidified with acetic acid, 26.32 cc. ’5 20

25

30

3.95 5.26 6.58 7.90

0.260 0.280 0,462 0.734

0.000 0.000 0 .

ooo

0.000

0.02

N per

,>

IOO

cc.

4.742

5.123

complete inversion

,,

,

2689

ADSORPTIOS BY ASH-FREE CHARCOAL

will retain when adsorbed on the charcoal. Insofar as this is true the method of preparation of the invertase may be expected to influence its adsorption behavior. Effect of Ignition of Charcoal on Adsorption of Invertase It is evident from the data presented in this paper that reproducible results can be obtained in the study of the adsorption behavior of invertase when ash-free charcoal is used as the adsorbent. I t should be emphasized, however, that since invertase prepared by the usual methods is colloidal in nature it is necessary rigidly to control the physical conditions involved in the adsorption experiments. I t was pointed out in the first part of this paper that the invertase is not the only variable in the adsorpt,ion experiments and that the adsorbent itself is far from completely defined. I t was stated as one of the conditions that the charcoal should be ignited just before use. The reason for this is as can be seen from Table XX that the adsorption of invertase by freshly ignited charcoal varies from that by charcoal which has not been freshly ignited. Freshly ignited charcoal adsorbs considerably larger quantities of invertase and the invertase is not so completely inactivated after adsorption. As a possible explanation for the difference it was thought that the nonignited charcoal had perhaps adsorbed larger amounts of gases, water vapour, etc. from the air. A few exploratory experiments, designed to test this hypothesis, such as impregnating the charcoal, after ignition, with oxygen or air and bubbling oxygen through the charcoal-invertase mixture during digestion were not productive of conclusive results.

TABLE XX Effect of Ignition of Charcoal on Adsorption of Invertase (Charcoal ignited for 2 0 minutes a t I O O O ~ C . ) Charcoal freshly ignited Grams invert Grams invert sugar found sugar found ase after digesafter digessolution tion of chartion of used coal carrying filtrate from adsorbed charcoal invertase Cc. invert-

Charcoal not freshly ignited Grams invert Grams invert Grams invert sugar Sound sugar found sugar proafter digesafter digesduced by tion of chartion of equal amount coal carrying filtrate of invertam adsorbed from charcoal in absence invertase of charcoal

Stock solution of invertase used I .

j

0.010

0.013 0.116 0 513 1.421

0.006 0.009 0.016 0.029

0.058

4.112

1.031 2.531 3 138

4.481 4.867 4.899

2 . 5

0.026 0.038

3.0

0.051

'5

0.000

0.000

0.000

0.435

20

0.007

0.000

I . 402

25

0 , 0 2 2

0.000

3 377

3.845 4.806 j . 091

30

0.032

0.262 I . 658 2.856

0.000

4.066

5 . '87

2 . 0

'

Diluted solution of invertase used

2690


Discussion It is generally conceded that little is definitely known concerning the chemical structure of enzymes and that more facts regarding their behavior are necessary before an answer can be given to the question of their structure. It is customary to state that enzymes are composed of a colloidal carrier and a n active group. I n this connection it may be well to quote a statement by Waldschmidt-Leitz,Y1“Willstatter considers therefore that the enzymes are composed of a colloidal bearer and a specific, active group, which enables them to be bound to the substrate, and the composition of which a t the same time conditions the colloidal nature of the entire complex. Such a concept gives equal value to the physico-chemical and to the structural chemical behavior of the enzymes. The problem of determining the specific material bearers of enzymic action suffers so much the less a loss in justification and significance, since it will be called on sooner or later to render a final decision between the various viewpoints as to the nature of enzymes.’, This statement reflects the status of our present knowledge of the structure of the enzyme complex. The existence of the colloidal carrier and that it is responsible for the colloidal behavior of the enzyme complex can hardly be questioned, but that the colloidal carrier is essential for the functioning of the specific group is not so certain since Willstatter has prepared invertase practically, if not ent,irely, free from yeast gum and concomitant colloidal substances. The enzyme thus freed, however, had lost much of its stability. Armstrong and Hilditch** also conclude from their consideration of the structure of the enzyme “moleculeJ’ that neither protein, carbohydrate, organic phosphorus, nor iron compounds is necessarily an integral component. As for the specific, active group, its structure, and manner of association with the colloidal carrier, there are few or no experimental facts on which an acceptable conjecture can be based. The data herein presented on the adsorption behavior of invertase afford further evidence of the colloidal nature of the invertase complex and the influence of various substances and conditions on its adsorption by ash-free charcoal. Possibly one of the most interesting and significant facts uncovered was that gum arabic added to an invertase preparation low in yeast gum content caused it t o take on the adsorption properties of a n invertase of high yeast gum content. Since the yeast gum and gum arabic presumably are of quite different origin and constitution and yet influence the behavior of the invertase in the same manner, and furthermore since Willstatter has shown that the complete separation of yeast gum from invertase leaves an active but unstable residue it seems a t least plausible that the invertase particle itself is of colloidal dimensions and that the yeast gum and gum arabic serve as protective colloids to stabilize it. The fact that the invertase can be freed from yeast gum would seem to make it unlikely that the active principle is chemically combined with the colloidal carrier. If the active part of the colloidal complex (yeast gum-invertase) is itself colloidal it would perhaps account for the fact that 2’

?3

E. Waldschmidt-Leitz: “Enzyme Actions and Properties”, 6 (1929). E. F. Armstrong and T. P. Hiiditch: J. Phys. Chem., 33, 1441 (1929).

ADSORPTIOX BY ASH-FREE CHARCOAL

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it is inactivated when adsorbed by charcoal and cannot be displaced or recovered from the charcoal, for it is well known that many substances in a colloidal state when agglomerated or coagulated lose their activity and cannot readily be redispersed. The specificity of the colloidal invertase for sucrose, for example, could be accounted for in the same way or rather would be no different from the specific action of many inorganic catalysts. If the “active group” is a definite chemical compound it would seem that small amounts a t least might be displaced from the charcoal and detected. It is hoped that the data presented in this paper have added something to the knowledge of the colloidal behavior of the invertase complex, a t least, as far as its adsorption by charcoal is concerned. Since mention has been made a t the beginning of this paper of the conclusions that have been drawn regarding the nature of invertase and its action from its adsorption by charcoal and behavior when thus adsorbed, it is unnecessary to repeat them here. It is sufficient to state that these conclusions were based on experiments which were carried out with charcoals of unknown purity and adsorptive properties. That impurities such as adsorbed acids and alkaline inorganic matter in the charcoals have marked effects on the adsorption of invertase and its behavior after adsorption need scarcely be mentioned in view of the results with the ash-free charcoal, and it is obvious that the results of the earlier works must be considered with these facts in mind. Attention should be called, however, to one or two points that have been raised by previous investigators, on which there are now additional facts to be taken into consideration. Nelson and Griffin9 raised the question of the possibility of an increased hydrogen-ion concentration on the charcoal surface as a factor influencing the adsorption and activity of the invertase. From the fact that acids adsorbed on charcoal are incapable of inverting sucrose it seems unlikely that there is a high concentration of hydrogen ions on the surface of the charcoal. D. Deutsch,22working in Freundlich’s laboratory has also produced evidence that in other systems, too, adsorption a t an interface results in a change of the adsorbed substance to the undissociated state. It would seem, therefore, that the question of a high hydrogen-ion concentration influencing the activity of the invertase on the charcoal can be answered in the negative. Nelson and Hitchcock12 as a result of their studies on the adsorption of invertase on charcoal stated that there was no evidence to indicate that the true activity of the invertase, as distinguished from resultant rate of hydrolysis of the sucrose in the mixture, was influenced by the combination of the invertase with the adsorbent. Willstatter has also shown that some preparations of invertase are as active when adsorbed on alumina as when free. From the fact that ash-free charcoal will adsorb relatively large amounts of invertase with resultant complete loss of ability to invert sucrose it seems certain that the combination of invertase with adsorbent must exert a profound influence on the activity of the invertase. It seems from the fact that it cannot be displaced that the invertase complex must indeed be destroyed.

** D. Deutsch:

Ber., 60B, 1036 (1927).

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ELROY J. MILLER AND SELMA L. BAXDEMER

The data presented in this paper indicate that much more must be known concerning the mechanism of adsorption of invertase by charcoal before farreaching conclusions on the nature of enzyme action can be drawn, as for example, the deduction by Michaelis’o that the law of mass action can be applied to the behavior of invertase adsorbed on charcoal. The results of this investigation also indicate that many of the seemingly contradictory results reported by previous investigators can be accounted for on the basis of adsorbed acids, alkaline impurities, etc., in the charcoal and the fact that the previous history of the charcoal and activity were not recognized as being important factors influencing the results. Summary The results of this investigation may be summarized as follows: I. A study has been made of the adsorption of invertase by ash-free adsorbent charcoal. Although invertase may be adsorbed on charcoal and lose its power of inverting sucrose just as do adsorbed acids, the two phenomena are quite different in other respects. 2. It has been found that, as in the case of adsorption of acids by charcoal, large amounts of invertase can be adsorbed with complete loss of ability to invert sucrose. The adsorption of still larger quantities of invertase results in the retention of some activity by the invertase. 3 . Attempts to displace and to reactivate adsorbed invertase were unsuccessful. This suggests that the invertase complex when adsorbed on charcoal undergoes decomposition with loss of inverting power. 4. The presence of acid in the invertase solution and of adsorbed acids on the charcoal enable the charcoal to adsorb much larger quantities of invertase from solution than are adsorbed when the charcoal is free from acid and the invertase solution neutral. 5. Invertase adsorbed from acid solution or by charcoal carrying adsorbed acids tends to retain some of its activity, in some cases as much as 50%. 6. The age of the invertase preparation, concentration, method of preparation, length of time of contact of charcoal with the invertase, presence of acids, and previous treatment of charcoal are factors which influence the adsorption behavior of invertase. 7 . I t has been found that gum arabic added to an invertase solution 10% in yeast gum content causes the invertase to take on the adsorption behavior of invertase of high yeast gum content. Since yeast gum and gum arabic have the same effect on the adsorption behavior it is suggested that this is additional evidence that they both function as a protective colloid for the invertase. 8. It has been suggested that the behavior of invertase in adsorption by charcoal can be accounted for on the assumption that the “active group” of the invertase is itself of colloidal dimensions. 9. I n the light of the results of this investigation the seemingly contradictory findings and conclusions of previously reported enzyme adsorption studies are acc0untc.d for and become of more value. East Lanszng, Mzchagan.

Adsorption from Solution by Ash-free Adsorbent Charcoal. VI - The

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