STUDIES ON PANCREATIC LIPASE

II. INFLUENCE OF VARIOUS COMPOUNDS ON TEE HYDROLYTIC ACTIVITY

BY S. 5. WEINSTEIN AND A. M. WYNNE

(From the Department of Biochemistry, University of Toronto, Toronto, Canada)

(Received for publication, September 25, 1935)

In the case of lipolytic enzymes practically nothing is known concerning the chemical nature of the groupings, in their mole- cules, which are associated with their peculiar activity. The precipitation experiments of Glick and King (1) suggest that either pancreatic lipase itself is a globulin or its activity is very intimately bound up with globulin. The work of Murray (2) indicates that the enzyme contains essential chemical groups which react with the carbonyl group of ketones.

The experiments to be reported in the present paper are con- cerned with the effects of various compounds on the activity of the enzyme and were undertaken with the hope that the results might lead to some further understanding of the nature of the reactive groups and assist in the formulation of a logical interpretation of lipase action.

Materials and Methods

The substances whose effects were investigated included ketones, aldehydes, cyanides, cyanate, thiocyanate, heavy metals, thiol compounds, quinone, polyhydric alcohols, halogen derivatives of fatty acids, halides, bile salts.

The degree of inhibition or of activation was calculated after determination of the initial velocity of hydrolysis of tripropionin in the presence and absence of the various substances. The enzyme preparation was a clarified 50 per cent of glycerol extract of pig pancreas powder; the experimental procedure for determin- ing the rate of hydrolysis was identical with that previously

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650 Studies on Pancreatic Lipase. II

described (3). The digestion mixtures were buffered at pH 8.9 with ammonia-ammonium chloride buffer; the temperature was 37”. Whenever necessary the solutions of the compounds were adjusted to the proper pH before addition to the enzyme solution. Prior to their addition to the other components of the digestion mixture the enzyme and the compound under investigation were allowed to remain in contact at 37” for varying periods of time. The enzyme to be added to control tubes was similarly treated with water equivalent in volume to that of the inhibitor solution employed. It was frequently observed that the degree of retarda- tion or of acceleration of the activity of the lipase was dependent upon the duration of this preliminary contact.

Each reaction tube contained the following substances: 30 cc. of COS-free distilled water, 2 cc. of CaC& solution (1 per cent), 5 cc. of egg albumin solution (2 per cent), 5 cc. of ammonia-ammo- nium chloride buffer, pH 8.95, 10 cc. of tripropionin emulsion, giving a concentration in the digestion mixture equivalent to 0.166 M, 2 cc. of enzyme solution containing the compound under investigation. In the experiments on the effects of the halides, calcium chloride was omitted as a standard constituent.

It should be stated that, in considering the data in Tables I to VIII which follow, inhibitions or activations of less than 5 per cent of the normal rate are regarded as being of doubtful significance. In each table the figures represent the relative initial velocities of hydrolysis effected by the enzyme treated with the various com- pounds, the results being expressed as percentages of the initial rate of hydrolysis obtained with untreated enzyme.

EXPERIMENTAL

Ketones and Related CompoundsIn confirmation of Murray’s findings, ketones are observed (Table I) to exercise marked inhib- itory effects, probably by attachment of the carbonyl group to reactive groups in the enzyme molecule. The degree of inhibition depends upon the time of preliminary contact between enzyme and inhibitor and is influenced by the molecular volume of the inhibitor, by the number of carbonyl groups present, and by the presence or absence of the benzene ring in the inhibitor molecule. Conversion of a ketone to the corresponding oxime (cf. ethyl methyl ketoxime) does not destroy the inhibitory capacity of the

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compound. The reactive forces in the oxime are probably the residual valencies of the nitrogen and oxygen atoms. The molec- ular volumes listed in Tables I and II were calculated from the data of Sugden (4).

Aldehydes-With the exception of paraldehyde, all aldehydes investigated caused marked inhibition of activity (Table II). The degree of inhibition increased with rising molecular volume in the series comprising formaldehyde, acetaldehyde, butyralde- hyde, and heptaldehyde; this progressive increase becomes appar-

TABLE I Influence of Ketones on Rate of Hydrolysis

Concentration of inhibitors in digestion mixtures, 3.7 X 10-a M.

Acetone ...................... Ethyl methyl ketone. ........ Diethyl ketone. ............ Dipropyl “ ............. Methyl n-hexyl ketone. ...... Acetyl acetone ............... Acetophenone ................ Cyclohexanone. .............. Cyclopentanone. ............. Ethyl methyl ketoxime .......

Time of preliminary contact of enzyme with inhibitor

- 105 100 100 100 53 0

42 74 86

100

- 75 00 74 79 28 39 2

58 50 60

- T

57 48 76 47 52 41 50 40 25 19 29 16 0 0

42 34 44 31 55 32

- - 45 37 37 33 40 29 25 15 14 12 2 0 0 0

14 7 25 19 24 10

161.5 198.2 236.2 240 260 207 310 249 212.5 228.8

* Calculated from the data of Sugden (4).

ent after 10 minutes preliminary contact with the enzyme. On the other hand paraldehyde, present in the same concentration, was very much less inhibitory, although its molecular volume is greater than that of heptaldehyde. The normally reactive carbonyl groups of the aldehydes are not free in paraldehyde, the oxygen atoms being components of the. ring. Aldol, containing a secondary alcohol group in addition to the aldehyde group, was more strongly inhibitory than butyraldehyde. Benzaldehyde was very strongly inhibitory, slightly more so than heptaldehyde, although their molecular volumes are almost identical. Benzene

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652 Studies on Pancreatic Lipase. II

itself was found to be inert when present in an amount equivalent to the concentration of the other substances tested. However, benzene is not sufficiently soluble in water to yield a solution of this concentration; to this fact one might attribute its apparent inertness, or it may be due to the absence of a reactive group in the molecule. On comparing the inhibitory capacities of heptal- dehyde and methyl n-hexyl ketone, compounds having approxi- mately equal molecular volumes, it is apparent that the aldehyde is much more toxic than the ketone.

It should be stated that the concentrations of the various com- pounds listed in Tables I to VIII represent the concentrations of

TABLE II Injluence of Aldehydes on Rate of Hydrolysis

Concentration of inhibitors in digestion mixtures, 3 X 10e3 M.

-~ Formaldehyde. .............. 111 86 75 72 61 54 82.2 Acetsldehyde ................ 92 84 64 50 34 24 121.2 Butyraldehyde. .............. 73 67 47 30 20 2 198 Aldol ........................ 65 43 36 2 0 0 208 Heptaldehyde. ............... 43 15 2 0 0 0 267 Paraldehyde ................. 97 97 97 97 93 87 279 Benzaldehyde. ............... 63 2 0 0 0 0 265

-

Time of contact I

the substances in the digestion mixtures. During preliminary contact with the enzyme each compound was present in the en- zyme solution in a concentration 27 times the recorded value.

Heavy MetalsIn Table III are summarized the results of experiments on the inhibitory powers of salts of heavy metals determined in the usual way after 15 minutes contact of enzyme with metallic compound. In contrast to their relative inertness, in concentrations of the order of 10ms M, toward the proteinase component of similar enzyme preparations (5), the salts of heavy metals exercised a marked inhibitory influence on the activity of the lipase when present in concentrations of the order of lo+ M. From the point of view of absolute values the figures in Table III

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are of doubtful significance, however, since the enzyme prepara- tions, like many others which have been subjected to studies of this kind, probably contained quite large amounts of impurities. Confirming the results of Platt and Dawson (6) and of Corran (7), copper was found to be more toxic than mercury in the same concentration. The opposite effect was obtained by Jacoby (8) for urease. Ferric sulfate was more toxic than the ferrous salt.

Cyanide, Cyanate, and Thiocyanate-Table IV shows the results obtained with these compounds. Both KCN and CH&N appeared to activate the lipase, the former more extensively than the latter. In each case the range of concentration associated with activation

TABLE III

Injluenee of Salts of Heavy Metals on Rate of Hydrolysis

Preliminary contact of enzyme with inhibitor, 15 minutes.

Salt concentration (X 10-s M)

0.5 1 1.0 ) 1.6 1 2.0

Rate of hydrolysin BB per cent of control rate

cuso 4. ........................... 85 54 42 HgClz ............................ 100 81 72 Fez(SO&. ........................ 91 78 51 FeSO, ............................ 100 100 83 coc12 ............................ 77 54 50

~-~ Colloidal iron*. .................. 93 93 76

* Concentration expressed in terms of molarity.

20 56 45 79 35

73

was quite narrow. Cyanate and thiocyanate were inert when used in the same concentrations as the cyanides. By plotting per cent activation against KCN concentration a smooth curve was obtained, showing maximum activation with about 6 X lo-” M

KCN. This, of course, is the concentration in the digestion mixture; in preliminary contact with enzyme, as previously stated, the’cyanide was present in 27 times this concentration. As shown in Table V it requires an appreciable time for the full influence of the cyanide on the enzyme to become manifest (in this experiment only after 15 minutes preliminary contact at 37”).

Activation by cyanide has previously been observed in the case of several other enzymes including papain, bromelin, cathepsin;

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654 Studies on Pancreatic Lipase. II

the subject has been reviewed by Grassmann (9), by Murray (lo), and by Tauber (11). Potato amylase was shown by Barker (12) to be susceptible to cyanide activation, and very recently Farber and Wynne (5) made a similar observation in the case of pancreatic proteinase.

TABLE IV Injeuence of Cyanide and Cyanatea on Rate of Hydrolyaia

Preliminary contact with enzyme, 15 minutee.

Concentration (X 1W u)

1 1 2.6 1 s 1 10 1 IS 1 20 1 a0 1 40 1 so 1 so0

Rata of hydrolysis aa per cant of control rate

Potaaeium cyanide.. . . . . .

TABLB V Influence of Duration of Preliminary Contact of Enzyme with Cyanide on

Degree of Activation Concentration of KCN in dkeetion mixture, 5 X 10-0 rd.

The of amteat Rate of hydrolysis m per cent of oontml rata

min. 0 5

10 15 30 00

100 120 130 160 149 150

Sulfur Compounds-In recent years several enzymes have been shown to be affected by the presence of thiol compounds; in some cases activation occurs. The influence of such compounds on enzyme activity has recently been reviewed by Bersin (13). Since pancreatic lipase has not, apparently, been investigated from this point of view, the experiments summarized in Table VI were carried out.

The enzyme and sulfur compound were allowed to remain in

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S. S. Weinstein and A. M. Wynne 655

preliminary contact for 60 minutes; otherwise the conditions were identical with those of previous experiments. Each of the com- pounds, cysteine, thioglycolic acid, and sodium hydrosulfite, caused definite activation when present in certain concentrations. Sodium sulfide and sodium bisulfite were inhibitory. The effects could not be attributed to change of pH since this was carefully controlled in all experiments.

The extent of the present work does not permit any conclusion as to the mechanism of lipase activation by thiol compounds. There is, however, no evidence to indicate that they exercise their effects by the reduction of toxic quinones, present as impurities, to dihydric phenols in the manner suggested by Quastel (14) to explain the protective action of thiol compounds upon urease in

TABLE VI Influence of Sulfur Compounds on Rate of Hydrolysis

Preliminary contact with enz ;Y me, 60 minutes.

Cysteine ....................... Thioglycolic acid ............... Sodium sulfide. ................

‘I hydrosulfite. .......... ‘I bisulfite. ..............

Concentration in digestion mixture (X 10-a ar)

1 1 2 1 3 1 4 1 6 1 6 1 7 1 10 1 25

Ftate of hydrolysis BS per cent of control rate

100 112 127 124 100 96 108 115

100 95 89 84 100 107 116 122

90 78 68 60

the presence of quinone or of compounds containing quinone as an impurity. It is probable that the amounts of quinone and related’ compounds in the enzyme preparations used in our experiments were negligible in view of the high solubility of these compounds in the solvents used in the treatment of the wet pancreas. Further- more, quinone in fairly high concentration has been shown to be only moderately inhibitory toward pancreatic lipase, very much less so than toward urease, which Quastel found to be 98 per cent inhibited by quinone in a dilution of 1: 2,500,OOO.

Phenol; Polyhydric Phenols, and Related CompoundsThe effects of several phenolic compounds on the activity of the enzyme after 60 minutes preliminary contact at 37” were examined. Four concentrations of each compound in the digestion mixture were

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Studies on Pancreatic Lipase. II

used; namely, 5, 7, 10, and 25 X low4 M; in preliminary contact with enzyme the compounds were present in concentrations 27 times these values. Included in the experiment were phenol, m- cresol, o-cresol, nitrophenol, catechol, resorcinol, hydroquinone, orcinol, guaiacol, phloroglucinol, pyrogallol, and inositol. Of these, only nitrophenol, catechol, resorcinol, hydroquinone, and orcinol had any appreciable influence, the degrees of inhibition by the highest concent,ration being 16, 12, 19, 33, and 20 per cent respectively. The inhibitory capacity of the dihydric phenols appears to increase with increased separation of the hydroxy groups. The presence of the methyl group, as in the cresols and orcinol, does not affect the toxicity of the parent compound; the nitro group, however, definitely increases the toxic properties of the phenol.

Orthoquinone-This compound, after 60 minutes contact with enzyme at 37”, caused inhibition of activity as follows, the concen- trations listed being those in the digestion mixtures.

Concentration Inhibition

x lo-’ Y 1 (About 1: 100,000) 2 3 4

I per cent

0 5

10 20

It was difficult to be certain of the results when concentrations of quinone higher than 5 X 1O-4 M were used, owing to color inter- ference with the titration end-point. When the results with inhibition (cj. Table I) by a substance such as acetophenone, in which the keto group is a part of the aliphatic chain, are compared, it is apparent that inclusion of the group in the aromatic ring reduces its effectiveness as an inhibitor.

Halogen Derivatives of Fatty Acid-The three compounds, chloro-, bromo-, and iodoacetic acid, inhibited the activity to an extent increasing in the order named (Table VII). Dickens (15) and Michaelis and Schubert (16) have suggested possible explanations of the mechanism of enzyme inhibition by these compounds. Dickens’ study of the speed of the reaction between glutathione and the three monohalogen derivatives of acetic acid

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S. S. Weinstein and A. M. Wynne 657

led him to conclude that the reaction is bimolecular and results in the liberation of the corresponding halide and the formation of a thio ether

R-SH + ICH&OOH = R-S-CHrCOOH + HI

Extending the consideration of this reaction to the inhibition of glyoxalase by monoiodoacetate, Dickens concluded that the inhibitor inactivated the enzyme system by combination with the coenzyme, glutathione. He found, furthermore, that iodo-, bromo-, and chloroacetic acids reacted with glutathione with relative speeds expressed by the ratio 15:9: 0.15. In the present work it was found that, when present in the concentration 1O-4 M, the three compounds inhibited lipase activity to an extent ex- pressed relatively by the figures 16, 11, and 4 for the iodo, bromo, and chloro acids respectively. There is, therefore, some resem- blance between the relative inhibitory capacities of the compounds on the one hand and the relative speeds of their reaction with glutathione on the other. Since it has been shown that sulfhydryl compounds activate the lipase, it is quite possible the halogen acetic acids may, at least partially, inactivate the enzyme by reacting with a natural sulfhydryl activator in the manner sug- gested by Dickens or with an essential -SH component of the enzyme molecule.

Michaelis and Schubert have, however, pointed out that iodo- acetic acid reacts quite easily with amino groups as follows:

RNHz + 2ICH&OONa + 2NaOH --t RN(CH&OONa)2 + 2NaI + 2HtO

and that the iodo acid is probably more reactive than the bromo and chloro compounds. In the case of the inhibition of enzymes by iodoacetic acid, Michaelis and Schubert have suggested that at pH 7 to 8 the point of attack of this acid may be considered as a -SH group provided this is confirmed by other evidence, but, “If the effect of iodoacetic acid occurs only at pH > 7 or 8, the point of attack may be just as well an amino group.”

From the evidence available it is impossible to decide whether reaction with the -SH group or the -NHz group or with both groups takes place during the inhibition of lipase activity by the halogen acetic acids. The susceptibility of the enzyme to inhibi- tion by aldehydes and heavy metals suggests that an amino group

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658 Studies on Pancreatic Lipase. II

may be an essential constituent of the enzyme; if so, the halogen acetic acids may quite possibly cause inactivation by union with this group.

Halides-Inhibition by these compounds increased in the order chloride, bromide, iodide, fluoride (Table VIII). With the excep- tion of fluoride these anions exercised effects in conformity with their positions in the lyotropic series; fluoride, huwever, was quite definitely the most toxic of all. Haldane’s suggestion (17) that

TABLE VII Influence of Monohalogen Derivatives of Acetic Acid on Rate of Hydrolysis

Preliminary contact with enzyme, 60 minutes.

Concentration in dig&ion mixture (X 10-a M)

1 ( 2.6 1 3 1 5 1 8 ( 10

Rate of hydrolysis as per cent of control rate

Chloroacetic acid.. . . . . . . . . . . . . . 96 Bromoacetic “ . . . . . . . . . . . . . . . . Iodoacetic “ . . . . . . . . . . . . . . . . /1oo1 g3 1 r: 1 ii 1 ii!

TABLE VIII Influence of Halides on Rate of Hydrolysis

Preliminary contact with enzyme, 60 minutes.

Concentration in digestion mixture (X 10-a M)

6 1 7 1 10 1 2s

Rate of hydrolysis aa per cent of control rate

Nacl............................. 96 89 81 73 NaBr.. . . . . . . . . . . . . . . . ._ . . . . . . . . . . 84 77 65 58 Nar.............................. 82 74 64 52 NaF.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 54 46 35

the inhibition of lipases by fluoride, observed by Rona and Pav- lovid (18), is due to its position in the Hofmeister series cannot, therefore, be regarded as offering a completely satisfactory expla- nation,

Bile Salts-WillstAtter and his collaborators (19, 20) observed that bile salts activated pancreatic lipase in an alkaline medium but caused inhibition in an acid medium. Glick and King (21) studied the effect of bile salts on the rate of hydrolysis of tributyrin

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by pancreatic lipase in a medium whose pH soon fell below 7.0 as the digestion proceeded. Both sodium taurocholate and glyco- cholate caused inhibition. In the presence of sodium oleate the rate of hydrolysis of emulsified olive oil by lipase was accelerated by both bile salts; the pH of the mixtures was not stated. The present experiments were carried out at pH 7.2 controlled by phosphate buffer; only initial velocities were used in calculating the relative rates. The tripropionin was emulsified by means of sodium oleate and the bile salts were in preliminary contact with the enzyme for 60 minutes at 37”. At approximate neutrality the bile salts, when present in the digestion mixtures in concentrations ranging from 0.5 to 4 X 1O-4 M, neither activated nor inhibited the enzyme.

DISCUSSION

The results as a whole show that the activity of the enzyme was affected by the presence of widely different chemical compounds. The ketones and aldehydes were definitely inhibitory when their reactive groups were free, indicating that the carbonyl group forms some sort of attachment with reactive groups in the enzyme mole- cule; the inhibitory capacities of closely related ketones and of closely related aldehydes bore a more or less direct relationship to the molecular volumes of the compounds. The evidence indi- cates that the inhibitory power of aldehydes is greater than that of related ketones having the same molecular volume. Heavy metals were inhibitory; cyanide activated the enzyme. The mechanism of this activation is not clear but preliminary experi- ments indicate that it is not entirely due to removal of toxic metals. Sulfhydryl compounds caused activation but apparently not, as in the case of urease, by reducing toxic quinones present as im- purity to the corresponding alcohols. Phenol and the cresols were relatively inert, but the presence of the nitro group increased the toxicity. The inhibitory capacity of the three dihydroxy- phenols increased with increased separation of the hydroxyl groups. Guaiacol, in which one of the -OH groups of catechol has been replaced by a -OCHa group was non-inhibitory, whereas catechol in the same concentration caused retardation. Neither of the two trihydroxy phenols investigated was inhibitory. Thus, of the hydroxy phenols, only the dihydroxy compounds were reactive.

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Monohalogen derivatives of acetic acid caused inhibition in the order I > Br > Cl. The significance of this observation has already been discussed in relation to the possible mechanism of the inhibition. The halogen anions were inhibitory in the order F > I > Br > Cl. Bile salts present in digestion mixtures buffered at approximate neutrality were without appreciable influence on the activity of the enzyme.

SUMMARY

The influence of various chemical compounds on the initial rate of hydrolysis of tripropionin by pancreatic lipase has been investi- gated. The compounds included ketones, aldehydes, salts of heavy metals, cyanides, cyanate, thiocyanate, thiol compounds, quinone, mono-, di-, and trihydric phenols, halogen derivatives of acetic acid, halides, and bile salts.

BIBLIOGRAPHY

1. Glick, D., and King, C. G., J. Am. Chem. Sot., 66,2445 (1933). 2. Murray, D. R. P., Biochem. J., 23,292 (1929). 3. Weinstein, S. S., and Wynne, A. M., J. Biol. Chem., 112,641 (1935-36). 4. Sugden, S., The parachor and valency, London (1930). 5. Farber, L., and Wynne, A.M., Biochem. J., 29,2323 (1935). 6. Platt, B. S., and Dawson, E. R., Biochem. J., 19,860 (1925). 7. Corran, R. F., Biochem. J., 23,133 (1929). 8. Jacoby, M., Biochem. Z., 269,211 (1933). 9. Grassmann, W., in Nord, F. F., and Weidenhagen, R., Ergebnisse der

Enzymforschung, Leipsic, 1, 152 (1932). 10. Murray, D. R. P., Biochem. J., 27,543 (1933). 11. Tauber, H., in Nord, F. F., and Weidenhagen, R., Ergebnisse der En-

zymforschung, Leipsic, 4,42 (1935). 12. Barker, J., Dept. SC. and Znd. Research, Rep. Food Znv. Board, 1 (1930). 13. Bersin, T., in Nord, F. F., and Weidenhagen, R., Ergebnisse der En-

zymforschung, Leipsic, 4, 63 (1935). 14. Quastel, J. H., Biochem. J., 27, 1116 (1933). 15. Dickens, F., Biochem. J., 27,114l (1933). 16. Michaelis, L., and Schubert, M. P., J. Biol. Chem., 166,331 (1934). 17. Haldane, J. B. S., Enzymes, Monographs on biochemistry, London

and New York (1930). 18. Rona, P., and Pavlovib, R., Biochem. Z., 134,108 (1923). 19. Willstatter, R., Waldschmidt-Leitz, E., and Memmen, F., 2. physiol.

Chem., 126,93 (1923). 20. Willstatter, R., and Memmen, F., Z. physiol. Chem., 133, 229 (1924). 21. Glick, D., and King, C. G., J. Biol. Chem., 97,675 (1932).

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S. S. Weinstein and A. M. WynneACTIVITY

COMPOUNDS ON THE HYDROLYTICINFLUENCE OF VARIOUS

STUDIES ON PANCREATIC LIPASE: II.

1936, 112:649-660.J. Biol. Chem. 

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