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Studies in bile salt solutions
S t u d i e s in Bile Salt S o l u t i o n s XVll. The Effect of Proteins on Bile-Salt-Stimulated Human Milk Lipase Activity against 4-Nitrophenylacetate 1 C H A R M I A N J. O ' C O N N O R
AND P E T E R W A L D E
Department of Chemistry, Universityof Auckland, Private Bag, Auckland, New Zealand Received May 29, 1985; accepted October 9, 1985 Bovine serum albumin, porcine pancreatic lipase, and horse heart myoglobin, but not any of bee venom melittin, human milk lysozyme,human milk lactotransferrin, human milk a-lactalbumin, human colostrum immunoglobulin A (IgA), or chicken egg white lysozyme, were found to catalyze the decomposition of 4-nitrophenylacetate (PNPA) in 0.1 M Tris, pH 7.5, 298 K, in the presence (2 raM) and absence of sodium taurocholate. The effect of each of these proteins (except bovine serum albumin) on the activity of bile salt-stimulated human milk lipase against PNPA was tested under the same conditions. In the absence of bile salt, human milk lysozyme had no effect, horse heart myoglobin was catalytic, and the other proteins were inhibitory. In the presence of bile salt, porcine pancreatic lipase and human milk lactotransferrin were slightly inhibitory, chicken egg white lysozyme was slightly catalytic, and the other proteins had no effect. A mechanism is proposed in which the bile salt competes with the enzyme for the protein interface. It is postulated that the binary bile salt-enzyme complex is active, whereas the binary protein-enzyme complex is not. © 1986AcademicPress,Inc.
INTRODUCTION Interfacial activation o f an e n z y m e can arise either because the substrate acquires new properties at the interface, or because the enz y m e itself is modified by absorption, or by a c o m b i n a t i o n o f these and other factors such as the increase o f substrate concentration at the interface. It is not possible to draw an unambiguous conclusion from activation studies on emulsified substrates since both the substrate and the e n z y m e will be affected by the presence o f the interface. A n understanding o f the m e c h a n i s m o f action o f enzymes on soluble substrates should help in elucidation o f the activating effect o f interfaces. A suitable system is, then, bile salt-stimulated h u m a n milk lipase and 4-nitrophenylacetate, P N P A . W e have recently shown (1) that a variety o f nonionic surfactants stimulate the hydrolysis Part XVl: O'Connor, c. J., and Walde, P., Langmuir 2, in press (1986).
reaction, but that ionic surfactants, with the exception o f the bile salts, behave as inactivators o f the esterase activity above a certain small concentration. The possible influence o f amphiphiles other than synthetic surfactants and bile salts o n the activity o f pancreatic lipase has been questioned by several authors (2-5). Activation by serum albumin, o f the hydrolysis o f t r i b u t y r i n catalysed by the enzyme, was attributed (2, 3) to prevention from surface denaturation o f the lipase by the protein adsorbed at the substratewater interface. Conversely, inhibition o f lipase activity by amphiphilic proteins appeared to be a general p h e n o m e n o n n o t directly related to a decrease in tension at the triacylglycerol-water interface and it was postulated that the inhibition could be the result o f desorption o f lipase from its substrate due to a change in interfacial quality. Like surfactants, proteins adsorb to oil-water interfaces (6) a n d cause a decrease in interfacial tension.
488 0021-9797/86 $3.00 Copyright © 1986 by Academic Press, lnc. All rights of reproduction in any form reserved.
Journal of Colloid and Interface Science, Vol, 112, No. 2, August 1986
STUDIES IN BILE SALT SOLUTIONS, XVII
The protein content of human milk is a relatively constant fraction of ca 0.9% (w/w) (7). Although most of these proteins are synthesized in the secretory cells of the mammary gland, breast milk also contains virtually all of the proteins found in the blood, albeit in small quantities. Ribadeau-Dumas et al. (8) have studied mammary proteins, and have shown that they are present in milk in two forms--micellar and soluble. The micellar form, which gives milk its white appearance, consists of spherical particles with a diameter varying from 10 to 100 nm, which incorporate particular proteins, the caseins and mineral ions (phosphate, calcium, and magnesium). The other proteins, known as "soluble proteins" are in true solution, and these include, in descending order of quantity (mg ml-1), o~lactalbumin, lactotransferrin, immunoglobulin A, serum albumin, and lysozyme. We have studied the effect of each of these proteins (except serum albumin) on the esterase activity of bile salt-stimulated milk lipase, both in the presence (2 mM) and absence of sodium taurocholate. In addition, we have been able to compare these results with those obtained in the presence of a number of other proteins, namely, porcine pancreatic lipase, chicken egg lysozyme, horse heart myoglobin, and bee venom melittin. This last protein is known to behave as a surfactant (9) because of the amphiphilic nature of its primary structure (10). EXPERIMENTAL
Materials. Sodium taurocholate (TC) was a product of Calbiochem; 4-nitrophenylacetate (PNPA), 2-amino 2-hydroxymethylpropane1,3-diol (Tris), chicken egg white lysozyme, alactalbumin, human colostrum IgA, and bovine serum albumin were products of Sigma; porcine pancreatic lipase was from Boehringer-Mannheim; and horse heart myoglobin was from Pentex. Melittin was isolated from bee venom by Dr. Barbara E. C. Banks, Department of Physiology, University College, London; lysozyme was isolated from human milk by Dr.
489
Peter M. Barling, Biochemistry Department, University of Auckland; and lactotransferrin was isolated from human milk by Dr. Sylvia V. Rumball, Department of Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North, and supplied as a solution (10 mg ml-J). Human milk lipase (HML) (a bile saltstimulated carboxyl-ester hydrolase EC 3.1.1.1) was purified from freshly collected milk according to the method described by Bl~ickberg and Hernell (11) using heparin Sepharose and reactive Blue 2-crosslinked agarose (Affigel Blue) supplied by Sigma. Activity of human milk lipase. The activity of HML was measured at 298 K using the soluble substrate 4-nitrophenylacetate with an initial concentration of 1.0 m M in Tris-HC1 buffer, 0.1 M, pH 7.5, either in the absence of bile salts or in the presence of 2 m M TC. The formation of the 4-nitrophenolate ion was followed at 400 nm (380 nm in the case of myoglobin) on either a Cary 15 or Cary 219 spectrophotometer and the measurements were corrected for the nonenzymatic hydrolysis of PNPA at pH 7.5. (The high absorption of myoglobin in the range greater than 400 nm necessitated the use of the shorter wavelength of 380 nm in the presence of this protein.)
Effect of different proteins on the activity of human milk lipase. A typical measurement was made as follows: x ml 0.1 M Tris-HC1 buffer, pH 7.5 (in the presence or absence of 2 mM TC) and y ml protein solution in 0.1 M Tris-HC1 buffer (in the presence or absence of 2 m M TC) were incubated at 298 K. The total volume (x + y) was 3 ml. The hydrolysis reaction was started by addition of 50 ~1 of substrate stock solution (PNPA, 60 m M in CH3CN) and 3 to 5 #1 of an enzyme stock solution (2 mg HML per ml H20) and the initial velocity of product formation was measured. The final solution contained 1.6% (v/ v) CH3CN. All measurements were corrected for the nonenzymatie hydrolysis of PNPA under identical conditions but in the absence of HML. The activity of HML (y = 0) in the presence or absence of 2 m M TC was set to Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
490
O'CONNOR
100% and the activity of H M L in the presence of the proteins was calculated relative to this value at zero protein concentration. It should be noted that in all cases with PNPA as substrate the activity at zero protein concentration was 6.5 times higher in the presence of TC than in its absence and this factor applies to all the experimental data presented herein. The concentration of protein was estimated spectrophotometrically at 280 nm by using known absorption coefficients. The values of E lclrl I~ and the maximum concentration of the proteins used in the kinetic studies were: bovine serum albumin 6.7(5), lO~tM;horse heart myoglobin 20.5(5), 10.3 ~M," melittin 16.4(5), 11.9 #M; human milk lysozyme 20.7 (calculated from a figure in Ref. (12)), 6.0 t~M, porcine pancreatic lipase 13.3(13), 2.2 #M; chicken egg white lysozyme 24.7(14), 21.9 ~tM; t~-lactalbumin from human milk 16.2(14), 7.0 ~zM;human colostrum IgA 12.4(14), 0.45 uM; human milk lactotransferrin, 2.1 ~tM.
Effect of different proteins on the nonenzymatic hydrolysis of 4-nitrophenylacetate. The experimental conditions were similar to those described above but the assays were carried out in the absence of HML. The reactions were initiated by addition of 50 ~tl of PNPA in CH3CN to 3 ml of buffered protein solution (in the presence or absence of taurocholate). The release ofp-nitrophenol in the absence of added protein was 1.4 + 0.1 ~tM min -1 and all initial rates were corrected for this spontaneous hydrolysis at pH 7.5. RESULTS
Esterase Activity of Proteins toward PNPA: Influence of Taurocholate The initial rate of hydrolysis of PNPA was determined in the presence of increasing concentrations of proteins and in both the presence and absence of TC at pH 7.5. Within the concentration range used, and in both the absence and presence of 2 m M TC, bee venom melittin, human milk lysozyme, human milk Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
AND
WALDE
lactotransferrin, chicken egg white lysozyme, human milk a-lactalbumin, and human colostrum IgA had no effect. The values for initial 4-nitrophenol released (#M rain -1) due to the presence of 1/~M of the remaining proteins (corrected for the spontaneous hydrolysis of PNPA at pH 7.5) were: bovine serum albumin 1.12 +_ 0.05; pancreatic lipase 0.53 +__0.01; and horse heart myoglobin 0.05 ___0.01. The increase in absorbance due to formation of the 4-nitrophenolate ion was linear with time in the presence of pancreatic lipase and horse heart myoglobin but in the presence of bovine serum albumin the increase was curved for the first 5 min and then became linear, indicating a two-step reaction mechanism including probably the formation of an acyl intermediate (15, 16). The extent of the catalysis seen in the presence of bovine serum albumin prevented study of its further effect on H M L activity against PNPA. If it be assumed that human serum albumin has a comparable effect with bovine serum albumin on the decomposition of PNPA, then it becomes of concern that the serum albumin might disturb the esterase activity measurements when whole milk ("contaminated" with serum albumin) is used as the source of human milk lipase. In our recent studies on the effect of storage on the esterase activity of H M L (17) we used up to 6/~1 of whole milk added to 3 ml of buffer and substrate. The concentration of serum albumin in human milk has been quoted as equal to 50 mg 100 ml -~ (18). At this concentration a maximum of only 1.6% of the total measured esterase activity could have been due to serum albumin activity. Initially, we found that the commercial sample of human colostrum IgA had a high and constant activity against PNPA in 0.1 M Tris-HC1 at pH 7.5 but we were able to confirm that this activity was attributable to contamination of the IgA by traces of HML. This contamination is not surprising since the concentration of IgA in human colostrum is ~ 17 mg m1-1 (19).
491
STUDIES IN BILE SALT SOLUTIONS, XVI1
The decomposition of monomeric PNPA by pancreatic lipase seems, at first sight, to be a contradiction in terms because it might not be expected that the lipase, which is well known for its ability to hydrolyze, at very high rates, emulsions of insoluble long-chain fatty acid esters of glycerol and other alcohols, should have activity against a substrate which is widely used for ordinary esterases and some proteases. However, it has been shown that the hydrolysis reaction of PNPA by pancreatic lipase in the presence of 4% CH3CN involves, as does that catalyzed by these other enzymes, an acylation and a deacylation step and that various interfaces could exert a considerable acceleration effect on the system (20). In all cases tested, the effect of the proteins on the rate of decomposition of PNPA at pH 7.5 was independent of whether, or not, 2 m M TC was present.
Activity of Human Milk Lipase in Presence of Proteins." Influence of Taurocholate The activity of human milk lipase against PNPA was determined in the presence of increasing concentrations of various proteins. The initial rate of hydrolysis was measured in both the absence and presence (2 mM) of TC. Results obtained with human milk lactotransferrin and human milk colostrum IgA are presented in Fig. 1 and those for human milk alactalbumin and human milk lysozyme are presented in Fig. 2. In the presence of TC, and under the conditions used, lactotransferrin had a relatively small and constant (at concentrations >0.1 t~M) inhibitory effect upon the activity, while human colostrum IgA, human milk a-lactalbumin and human milk lysozyme had no effect. In the absence of TC, and under the conditions used, human milk lysozyme had no effect, human milk a-lactalbumin had a small, but significant inhibitory effect (at concentrations of added protein > 1 ~Mthere was a 25% inhibition), while human milk lactotransferrin
2001 RELATIVE ACTIVITY
(~)
I
oi o
10
O
2ooI RELATIVE / ACTIVITY [ I~)
i A looT. :
c
0.0
.
02
0.4
0.g
0.8
1.0
1.2
,;c_~-
2,0
[PROTEIN] (pM)
FIG. 1. Variation of the relative initial rate of human milk lipase-catalyzed decomposition of 4-nitrophenylacetate in 0.1 M Tris/HCl buffer containing 1.6% (v/v) acetonitrile at pH 7.5, 25°C, in the presence of (O, O) human milk lactotransferrin, (~, a) human colostrum IgA. Open symbols, in the absence of taurocholate; closed symbols, in the presence (2 mM) of taurocholate.
and human colostrum IgA were strong inhibitors and almost equal in their effect. As little as ~0.2 tzM human lactotransferrin present in the assay mixture caused a 70% inhibition of the HML activity, compared with the activity in the absence of lactotransferrin. This level remains constant as the concentration of lactotransferrin was increased to 0.8 uM but, at 2 tsM, 85% inhibition was observed. Results obtained with horse heart myoglobin, bee venom melittin and chicken egg white lysozyme are presented in Fig. 3 and those for porcine pancreatic lipase in Fig. 4. Under the conditions used, myoglobin (< 10 /zM) increased the esterase activity of HML in the absence of TC, whereas melittin (<12 #M) and chicken egg white lysozyme (<22 ~tM) decreased the activity to ca. 50%. This activity was achieved in the presence of 0.4 #M melittin or chicken lysozyme and at higher concentrations the activity remained almost constant at this depressed level. The activity of HML against PNPA (in the absence of bile salt) was completely inhibited, Journal of Colloid and Interface Science, Vol.
112, N o . 2, A u g u s t 1986
492
O'CONNOR AND WALDE 2ooI
hydrolytic enzymes are able to catalyze the decomposition of ester substrates. Sperm whale metmyoglobin (21), bovine mercaptalb u m i n (15), and h u m a n serum albumin (16, (~) 22) catalyze, for example, the decomposition of PNPA, a c o m p o u n d which is subjected to general acid-general base catalysis in both aqueous (23) and nonaqueous solutions (24). ~oo1 RELATIVE / In the present work we have found that ACTIVITY a m o n g the proteins tested bovine serum al[ ~ ~00[ b u m i n was the most effective in releasing pnitrophenol from the water soluble substrate PNPA. Due to this high activity the effect of 0 i 2 3 L s 6~ a serum albumin on the activity of bile salt[PROTEIN]()~M) stimulated lipase could not be studied. In the FIG. 2. Variation of the relative initial rate of human other cases, no biphasic reaction was observed milk lipase-eatalyzed decomposition of 4-nitrophenyl- and the extent, if any, of the catalysis induced acetate in 0.1 M Tris/HC1 buffer containing 1.6% (v/v) by the presence of added protein seems to be acetonitrile at pH 7.5, 25°C, in the presence of (©, O) human milk a-lactalbumin, (A, A) human milk lysozyme. related to the n u m b e r of imidazole groups (histidine residues) of the protein molecule Open and closed symbolsas in Fig. 1. RELATtVE [ ACTIVITY
~OOT: Ao
o
o
o
o
under the conditions used, by the presence of 1 ~tM pancreatic lipase. Even in the presence of 0.2 u M pancreatic lipase the activity of H M L was ~ 5-10% compared with the H M L activity in the absence of protein. Under the conditions used, neither myoglobin nor melittin had any effect on the activity of H M L in the presence of TC. Porcine pancreatic lipase had a small inhibitory effect (30% in the presence of 1 ~ M protein) compared with the activity in the absence of pancreatic lipase, while chicken egg white lysozyme had a small catalytic effect (130% in the presence of 8 # M protein). Further experiments were performed to show that t h e effect of pancreatic lipase u p o n H M L is reversible; inhibition can be reversed by adding TC after addition of pancreatic lipase to HML. The incubations were carried out both in the absence of CH3CN and substrate (PNPA) and in their presence. DISCUSSION It has previously been reported that several proteins which are not normally classified as Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
COO 13 [3
300 RELATIVE ACTIVITY (%) 2O0
0
0
1001 o
&
a~
,y
2°° I
RELATIVE ACTIVITy
:,
(%) 100~ •
0
•
2
~
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I
6
8
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4/-~-12 22 2t,
[PROTEIN](pM)
FIG. 3. Variation of the relative initial rate of human milk lipase catalyzed decomposition of 4-nitrophenylacetate in 0.1 M Tris/HCl buffercontaining 1.6%(v/v) acetonitrile at pH 7.5, 25°C, in the presenceof(O, g) chicken egg white lysozyme,(A, A) melittin from bee venom, (D, u) horse heart myoglobin.Open and closed symbolsas in Fig. 1.
STUDIES IN BILE SALT SOLUTIONS, XVII
493
200t
was little affected by these steroidal surfactants. In the light of this result, it is not surprising ACTIVfTY (%) that there was an absence of any additional 100 effect, in the presence of TC, in these present o experiments. o _ _ Q r, _ _ ,~ It has repeatedly been reported that proteins "unfold" at the interfaces which separate a polar from an apolar phase (26). The majority 2oo1 RELATIVE ] of the polar groups of native globular proteins ACTIVITY ~ • c~> ; " in aqueous solution are now known to be at the surface of the molecules in contact with the solvent, while most of the hydrophobic groups are buried inside where they ensure in0.2 0.3 0J~ 05 0.6 1.0 1.1 12 0.0 0.1 ternal stability. Therefore the proximity of an [PROTEIN] (pt4) amphipathic compound may be expected to FIG. 4. Variation of the relative initial rate of human alter several groups in the protein and thus milk lipase catalyzed decomposition of 4-nitrophenylmodify its three-dimensional structure. The acetate in 0.1 M Tris/HC1 buffer containing 1.6% (v/v) acetonitrile at pH 7.5, 25°C, in the presence of porcine degree of surface denaturation may depend on pancreatic lipase. Open and closed symbols as in Fig. 1. the hydrophobicity of the amphiphile which is adsorbed, and in the situation when the protein is an enzyme, interfacial adsorption may (and in particular the number of histidine res- induce progressive inactivation. idues lying on the surface of the molecule). The effect on the activity of human milk Thus, bovine serum albumin, with 17 histidine lipase by human lactotransferrin, human coresidues, is strongly catalytic, porcine pan- lostrum IgA, human milk oMactalbumin, bee creatic lipase (10 histidine residues) and myo- venom melittin, horse heart myoglobin, and globin (11 residues) are moderately catalytic, porcine pancreatic lipase can, in each case, be while human lactotransferrin (8 residues), hu- accounted for by postulating sorption of these man milk lysozyme, chicken egg white lyso- proteins on the surface of the enzyme. The zyme, and human milk a-lactalbumin (each magnitude of the inhibition will be related to with 1 residue) and melittin (zero histidine the capacity of the enzyme to interact with the residues) are ineffective. Breslow and Gurd protein. The absence of any effect in the pres(21) have shown that 6 imidazole groups of ence of human milk lysozyme indicates either native metmyoglobin are freely reactive and that there is insignificant affinity o f the enzyme responsible for the catalytic decomposition of for this protein or that, if sorption does occur, PNPA by this protein. Although human lac- it does not have an effect on the rate of cataltotransferrin has 8 histidine residues per mol- ysis. In each of the cases in which inhibition was ecule, under the conditions used no catalytic effect on the hydrolysis of PNPA could be ob- observed in the absence of bile salt there was served. This result indicates that at least some an absolute requirement of bile salt to relieve of these imidazole groups may lie buried be- inhibition of the lipase system by protein. The neath the protein surface or are less reactive, concentration of bile salt used, 2 mM, could not fully relieve the inhibition in the case o f due to local environmental effects. O'Connor and Wallace (25) studied the ef- porcine pancreatic lipase and human milk fect of a range of bile salts on the nonenzy- lactotransferrin. It appears that the bile salt competes with matic hydrolysis of PNPA and other esters and found that the stability of these compounds the enzyme for the protein interface and, in a RELATIVE
ioo~.
I
•
Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
494
O'CONNOR AND WALDE
concentration above its critical micelle con- of bile salt-stimulated lipase is achieved by the centration, it clears the enzyme interface of monomeric bile salt (28). Certainly, in the case protein. A specific reunion of the unmodified of the cholates, activity increases with increasenzyme with the substrate, and a restoration ing bile salt concentration up to the critical of catalytic activity is affected in the presence micelle concentration and then remains at the of bile salt. The restoration of a constant ac- constant level maintained by the equilibrium tivity, even in the presence of excess protein, concentration of bile salt monomer. Such an is an effect of the physical separation of protein effect of bile salt in separating the protein from from the enzyme. A possible reaction scheme the enzyme is a general surfactant effect and is given in Fig. 5. The reversible inactivation supports the conclusion that lipase inactivaof the enzymic activity, noted in the presence tion is not related directly to the change in of pancreatic lipase, supports this scheme interracial tension brought about by the prowhich describes the formation of a bile salt tein but to desorption of lipase from its submicelle-protein complex which does not in- strate. teract with the enzyme. Such a postulate is The activation of the activity of human milk consistent with the overwhelming evidence lipase by horse heart myoglobin and the rethat the modification of pancreatic porcine li- versal of this activation in the presence of bile pase activity by bile salts is linked to a variation salt is not inconsistent with the above discusin the adsorption capacity of the enzyme aris- sion. We have already suggested that nonionic ing from a modification of the interface (27). surfactants interact with the enzyme and We have previously suggested that activation thereby induce a conformational change which
+ S '-
NO ESTERASE ACTIVITY
ESTERASE ACTIVITY
FIG. 5. Tentative scheme for the anchoring of h u m a n milk lipase, L, by protein, P, and for reversal of deactivation in the presence of an equilibrium mixture of bile salt micelles and monomers. S is an ester substrate. The molecules are not drawn to scale. Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
STUDIES IN BILE SALT SOLUTIONS, XVII
leads to an increase in the binding of the substrate to the enzyme (1). In a similar way, we can postulate that the interaction ofmyoglobin with human milk lipase causes a conformational change which is favorable for enzymatic activity, but that the presence of bile salt separates the protein from the enzyme, thereby restoring the original conformation and the activity observed in the absence of protein. It might have been assumed that the magnitude of the interaction between the protein and the enzyme, and thereby the degree of catalysis and inhibition, was related to the hydrophobicity of the protein. A method has been devised for calculating the average hydrophobicity of proteins, H~av, from their amino acid composition, without taking into account possible contributions caused by the secondary structure of the protein (29). A number of alternative hydrophobicity scales has been constructed simply by comparing the ratio (R) of the frequencies of occurrence (~) of whatever particular side chain one wishes to stress. One feature of proteins that is fairly reliable is the tendency of charged or very polar residues to be external. A particular ratio scale (R3) is constructed from
R3-
k
[1]
J
where k represents the polar side chains (Lys + Arg + His + AsX + G1X), j represents apolar side chains (Ile + Tyr + Phe + Leu + Val + Met), and ~pis the frequency of occurrence ofk orj per mole protein (30, 31). This choice of residues is arbitrary, but convenient, since it spreads out different proteins over a relatively wide scale. R3 and H,av show some anticorrelation, but they are by no means purely inverse measures. R3 allows a fairly clear distinction between internal and external membrane proteins, whereas H~av does not. We calculated values of H~avand R3 for the proteins which have been used to modify the
495
activity of HML in this study, but we found no relationship between enzymatic activity and those values. This result gives further support to the conclusion that the activity is not related to the surface tension of the protein. We also compared the activity with the relative values of the isoelectric points of the proteins but found that the activity (or lack of it) did not seem to be directly related to the acidity of the surface of the protein. ACKNOWLEDGMENTS Equipment grants from the Research Committees of the Universities of New Zealand Grants' Committee and the University of Auckland and the award of a University of Auckland Postdoctoral Fellowship (to PW) are gratefully acknowledged. We are indebted to the staff of National Women's Hospital, Auckland, for collecting the breast milk; to Dr. B. E. C. Banks, Dr. P. M. Barling, and Dr. S. V. Rumball for the kind donation of chemicals; to the members of the Biochemistry Department, University of Auckland, for assistance in purification of the enzyme; and to the New Zealand Foundation for the Newborn for financial assistance. REFERENCES 1. O'Connor, C. J., and Walde, P., Langmuir, 2, in press (1986); O'Connor, C. J., Stockley, I. C., and Walde, P., N.Z. Med. J. 98, 306 (1985). 2. Brockerhoff, H., J. Biol. Chem. 246, 5828 (1971). 3. Borgstrrm, B., and Erlanson, C., Gastroenterology 75, 382 (1978). 4. Bl~ickberg, L., Hernell, O., Bengtsson, G., and Olivecrona, T., J. Clin. Invest. 64, 1303 (1979). 5. Gargouri, Y., Julien, R., Sugihara, A., Verger, R., and Sarda, L., Biochim. Biophys. Acta 795, 326 (1984). 6. Macritchie, F., Adv. Protein Chem. 37, 283 (1978). 7. Jensen, A. A., in "Residue Reviews" (F. A. Gunther and J. D. Gunther, Eds.), Vol. 83, pp. 2-128. Springer-Verlag, New York, 1983. 8. Ribadeau-Dumas, B., Endeavour 7, 80 (1983). 9. Habermann, E., Science 177, 314 (1972). 10. Habermann, E., and Jentsch, J., Hoppe-Seyler's Z. Physiol. Chem. 348, 37 0967). I I. Bl~ickberg,L., and Hernell, O., Eur. J. Biochem. 116, 221 (1981). 12. Jolles, J., and Jolles, P., Biochemistry 6, 411 (1967). 13. Desnuelle, P., in "The Enzymes" (P. D. Boyer, Ed.), 3rd ed., Vol. VII, pp. 575-616. Academic Press, New York, 1972. 14. Fasman, G. D., Ed., in "C. R. C. Handbook of BioJournal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
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chemistry and Molecular Biology Proteins, 3rd ed.," Vol. II, pp. 383-545, C. R. C. Press, Boca Raton, Fla., 1976. 15. Tildon, J. T., and Ogilvie, J. W., J. Biol. Chem. 247, 1265 (1972). 16. Means, G, E., and Bender, M. L., Biochemistry 14, 4989 (1975). 17. O'Connor, C. J., and Walde, P., N.Z. Med. J. 98, 114 (1985). 18. Blanc, B., WorldRev. Nutr. Diet36, 1 (1981). 19. Bezkoravalny, A., J. Dairy Sci. 60, 1023 (1977). 20. S~m~riva, M., Chapus, C., Bovier-Lapierre, C., and Desnuelle, P., Biochem. Biophys. Res. Commun. 58, 808 (1974). 21. Breslow, E., and Gurd, F. R. N. J. Biol. Chem. 237, 371 (1962). 22. Kurono, Y., Maki, T., Yotsuyanagi, T., and Ikeda, K., Chem. Pharm. Bull. 27, 2781 (1979). 23. O'Connor, C. J., and Wallace, R. G., Aust. J. Chem 37, 2559 (1984).
Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
24. O'Connor, C. J., Lomax, T. D., and Ramage, R. E., Adv. Colloid Interface Sci. 20, 21 (1984). 25. O'Connor, C. J., and Wallace, R. G., Aust. J. Chem. 37, 1881 (1984). 26. James, L. K., and Augenstein, L., Adv. Enzymol. 28, 1 (1966). 27. Vandermeers, A., Vanderrneers-Piret, M. C., Rathe, J., and Christophe, J., Biochem. Biophys. Res. Commun. 69, 790 (1976). 28. O'Connor, C. J., and Wallace, R. G., £ Colloid Interface Sci. 102, 539 (1984). 29. Fasman, G. D., Ed., in "C. R. C. Handbook of Biochemistry and Molecular Biology, Proteins, 3rd ed.," Vol. I, pp. 209-210. C. R. C. Press, Boca Raton, Fla., 1976. 30. Cantor, C. R., and Schimmel, P. R., "Biophysical Chemistry," Part I, pp. 55ff. Freeman, San Francisco, 1980. 31. Barrantes, F. J., Biochem. Biophys. Res. Commun. 62, 407 (1975).
AND P E T E R W A L D E
Department of Chemistry, Universityof Auckland, Private Bag, Auckland, New Zealand Received May 29, 1985; accepted October 9, 1985 Bovine serum albumin, porcine pancreatic lipase, and horse heart myoglobin, but not any of bee venom melittin, human milk lysozyme,human milk lactotransferrin, human milk a-lactalbumin, human colostrum immunoglobulin A (IgA), or chicken egg white lysozyme, were found to catalyze the decomposition of 4-nitrophenylacetate (PNPA) in 0.1 M Tris, pH 7.5, 298 K, in the presence (2 raM) and absence of sodium taurocholate. The effect of each of these proteins (except bovine serum albumin) on the activity of bile salt-stimulated human milk lipase against PNPA was tested under the same conditions. In the absence of bile salt, human milk lysozyme had no effect, horse heart myoglobin was catalytic, and the other proteins were inhibitory. In the presence of bile salt, porcine pancreatic lipase and human milk lactotransferrin were slightly inhibitory, chicken egg white lysozyme was slightly catalytic, and the other proteins had no effect. A mechanism is proposed in which the bile salt competes with the enzyme for the protein interface. It is postulated that the binary bile salt-enzyme complex is active, whereas the binary protein-enzyme complex is not. © 1986AcademicPress,Inc.
INTRODUCTION Interfacial activation o f an e n z y m e can arise either because the substrate acquires new properties at the interface, or because the enz y m e itself is modified by absorption, or by a c o m b i n a t i o n o f these and other factors such as the increase o f substrate concentration at the interface. It is not possible to draw an unambiguous conclusion from activation studies on emulsified substrates since both the substrate and the e n z y m e will be affected by the presence o f the interface. A n understanding o f the m e c h a n i s m o f action o f enzymes on soluble substrates should help in elucidation o f the activating effect o f interfaces. A suitable system is, then, bile salt-stimulated h u m a n milk lipase and 4-nitrophenylacetate, P N P A . W e have recently shown (1) that a variety o f nonionic surfactants stimulate the hydrolysis Part XVl: O'Connor, c. J., and Walde, P., Langmuir 2, in press (1986).
reaction, but that ionic surfactants, with the exception o f the bile salts, behave as inactivators o f the esterase activity above a certain small concentration. The possible influence o f amphiphiles other than synthetic surfactants and bile salts o n the activity o f pancreatic lipase has been questioned by several authors (2-5). Activation by serum albumin, o f the hydrolysis o f t r i b u t y r i n catalysed by the enzyme, was attributed (2, 3) to prevention from surface denaturation o f the lipase by the protein adsorbed at the substratewater interface. Conversely, inhibition o f lipase activity by amphiphilic proteins appeared to be a general p h e n o m e n o n n o t directly related to a decrease in tension at the triacylglycerol-water interface and it was postulated that the inhibition could be the result o f desorption o f lipase from its substrate due to a change in interfacial quality. Like surfactants, proteins adsorb to oil-water interfaces (6) a n d cause a decrease in interfacial tension.
488 0021-9797/86 $3.00 Copyright © 1986 by Academic Press, lnc. All rights of reproduction in any form reserved.
Journal of Colloid and Interface Science, Vol, 112, No. 2, August 1986
STUDIES IN BILE SALT SOLUTIONS, XVII
The protein content of human milk is a relatively constant fraction of ca 0.9% (w/w) (7). Although most of these proteins are synthesized in the secretory cells of the mammary gland, breast milk also contains virtually all of the proteins found in the blood, albeit in small quantities. Ribadeau-Dumas et al. (8) have studied mammary proteins, and have shown that they are present in milk in two forms--micellar and soluble. The micellar form, which gives milk its white appearance, consists of spherical particles with a diameter varying from 10 to 100 nm, which incorporate particular proteins, the caseins and mineral ions (phosphate, calcium, and magnesium). The other proteins, known as "soluble proteins" are in true solution, and these include, in descending order of quantity (mg ml-1), o~lactalbumin, lactotransferrin, immunoglobulin A, serum albumin, and lysozyme. We have studied the effect of each of these proteins (except serum albumin) on the esterase activity of bile salt-stimulated milk lipase, both in the presence (2 mM) and absence of sodium taurocholate. In addition, we have been able to compare these results with those obtained in the presence of a number of other proteins, namely, porcine pancreatic lipase, chicken egg lysozyme, horse heart myoglobin, and bee venom melittin. This last protein is known to behave as a surfactant (9) because of the amphiphilic nature of its primary structure (10). EXPERIMENTAL
Materials. Sodium taurocholate (TC) was a product of Calbiochem; 4-nitrophenylacetate (PNPA), 2-amino 2-hydroxymethylpropane1,3-diol (Tris), chicken egg white lysozyme, alactalbumin, human colostrum IgA, and bovine serum albumin were products of Sigma; porcine pancreatic lipase was from Boehringer-Mannheim; and horse heart myoglobin was from Pentex. Melittin was isolated from bee venom by Dr. Barbara E. C. Banks, Department of Physiology, University College, London; lysozyme was isolated from human milk by Dr.
489
Peter M. Barling, Biochemistry Department, University of Auckland; and lactotransferrin was isolated from human milk by Dr. Sylvia V. Rumball, Department of Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North, and supplied as a solution (10 mg ml-J). Human milk lipase (HML) (a bile saltstimulated carboxyl-ester hydrolase EC 3.1.1.1) was purified from freshly collected milk according to the method described by Bl~ickberg and Hernell (11) using heparin Sepharose and reactive Blue 2-crosslinked agarose (Affigel Blue) supplied by Sigma. Activity of human milk lipase. The activity of HML was measured at 298 K using the soluble substrate 4-nitrophenylacetate with an initial concentration of 1.0 m M in Tris-HC1 buffer, 0.1 M, pH 7.5, either in the absence of bile salts or in the presence of 2 m M TC. The formation of the 4-nitrophenolate ion was followed at 400 nm (380 nm in the case of myoglobin) on either a Cary 15 or Cary 219 spectrophotometer and the measurements were corrected for the nonenzymatic hydrolysis of PNPA at pH 7.5. (The high absorption of myoglobin in the range greater than 400 nm necessitated the use of the shorter wavelength of 380 nm in the presence of this protein.)
Effect of different proteins on the activity of human milk lipase. A typical measurement was made as follows: x ml 0.1 M Tris-HC1 buffer, pH 7.5 (in the presence or absence of 2 mM TC) and y ml protein solution in 0.1 M Tris-HC1 buffer (in the presence or absence of 2 m M TC) were incubated at 298 K. The total volume (x + y) was 3 ml. The hydrolysis reaction was started by addition of 50 ~1 of substrate stock solution (PNPA, 60 m M in CH3CN) and 3 to 5 #1 of an enzyme stock solution (2 mg HML per ml H20) and the initial velocity of product formation was measured. The final solution contained 1.6% (v/ v) CH3CN. All measurements were corrected for the nonenzymatie hydrolysis of PNPA under identical conditions but in the absence of HML. The activity of HML (y = 0) in the presence or absence of 2 m M TC was set to Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
490
O'CONNOR
100% and the activity of H M L in the presence of the proteins was calculated relative to this value at zero protein concentration. It should be noted that in all cases with PNPA as substrate the activity at zero protein concentration was 6.5 times higher in the presence of TC than in its absence and this factor applies to all the experimental data presented herein. The concentration of protein was estimated spectrophotometrically at 280 nm by using known absorption coefficients. The values of E lclrl I~ and the maximum concentration of the proteins used in the kinetic studies were: bovine serum albumin 6.7(5), lO~tM;horse heart myoglobin 20.5(5), 10.3 ~M," melittin 16.4(5), 11.9 #M; human milk lysozyme 20.7 (calculated from a figure in Ref. (12)), 6.0 t~M, porcine pancreatic lipase 13.3(13), 2.2 #M; chicken egg white lysozyme 24.7(14), 21.9 ~tM; t~-lactalbumin from human milk 16.2(14), 7.0 ~zM;human colostrum IgA 12.4(14), 0.45 uM; human milk lactotransferrin, 2.1 ~tM.
Effect of different proteins on the nonenzymatic hydrolysis of 4-nitrophenylacetate. The experimental conditions were similar to those described above but the assays were carried out in the absence of HML. The reactions were initiated by addition of 50 ~tl of PNPA in CH3CN to 3 ml of buffered protein solution (in the presence or absence of taurocholate). The release ofp-nitrophenol in the absence of added protein was 1.4 + 0.1 ~tM min -1 and all initial rates were corrected for this spontaneous hydrolysis at pH 7.5. RESULTS
Esterase Activity of Proteins toward PNPA: Influence of Taurocholate The initial rate of hydrolysis of PNPA was determined in the presence of increasing concentrations of proteins and in both the presence and absence of TC at pH 7.5. Within the concentration range used, and in both the absence and presence of 2 m M TC, bee venom melittin, human milk lysozyme, human milk Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
AND
WALDE
lactotransferrin, chicken egg white lysozyme, human milk a-lactalbumin, and human colostrum IgA had no effect. The values for initial 4-nitrophenol released (#M rain -1) due to the presence of 1/~M of the remaining proteins (corrected for the spontaneous hydrolysis of PNPA at pH 7.5) were: bovine serum albumin 1.12 +_ 0.05; pancreatic lipase 0.53 +__0.01; and horse heart myoglobin 0.05 ___0.01. The increase in absorbance due to formation of the 4-nitrophenolate ion was linear with time in the presence of pancreatic lipase and horse heart myoglobin but in the presence of bovine serum albumin the increase was curved for the first 5 min and then became linear, indicating a two-step reaction mechanism including probably the formation of an acyl intermediate (15, 16). The extent of the catalysis seen in the presence of bovine serum albumin prevented study of its further effect on H M L activity against PNPA. If it be assumed that human serum albumin has a comparable effect with bovine serum albumin on the decomposition of PNPA, then it becomes of concern that the serum albumin might disturb the esterase activity measurements when whole milk ("contaminated" with serum albumin) is used as the source of human milk lipase. In our recent studies on the effect of storage on the esterase activity of H M L (17) we used up to 6/~1 of whole milk added to 3 ml of buffer and substrate. The concentration of serum albumin in human milk has been quoted as equal to 50 mg 100 ml -~ (18). At this concentration a maximum of only 1.6% of the total measured esterase activity could have been due to serum albumin activity. Initially, we found that the commercial sample of human colostrum IgA had a high and constant activity against PNPA in 0.1 M Tris-HC1 at pH 7.5 but we were able to confirm that this activity was attributable to contamination of the IgA by traces of HML. This contamination is not surprising since the concentration of IgA in human colostrum is ~ 17 mg m1-1 (19).
491
STUDIES IN BILE SALT SOLUTIONS, XVI1
The decomposition of monomeric PNPA by pancreatic lipase seems, at first sight, to be a contradiction in terms because it might not be expected that the lipase, which is well known for its ability to hydrolyze, at very high rates, emulsions of insoluble long-chain fatty acid esters of glycerol and other alcohols, should have activity against a substrate which is widely used for ordinary esterases and some proteases. However, it has been shown that the hydrolysis reaction of PNPA by pancreatic lipase in the presence of 4% CH3CN involves, as does that catalyzed by these other enzymes, an acylation and a deacylation step and that various interfaces could exert a considerable acceleration effect on the system (20). In all cases tested, the effect of the proteins on the rate of decomposition of PNPA at pH 7.5 was independent of whether, or not, 2 m M TC was present.
Activity of Human Milk Lipase in Presence of Proteins." Influence of Taurocholate The activity of human milk lipase against PNPA was determined in the presence of increasing concentrations of various proteins. The initial rate of hydrolysis was measured in both the absence and presence (2 mM) of TC. Results obtained with human milk lactotransferrin and human milk colostrum IgA are presented in Fig. 1 and those for human milk alactalbumin and human milk lysozyme are presented in Fig. 2. In the presence of TC, and under the conditions used, lactotransferrin had a relatively small and constant (at concentrations >0.1 t~M) inhibitory effect upon the activity, while human colostrum IgA, human milk a-lactalbumin and human milk lysozyme had no effect. In the absence of TC, and under the conditions used, human milk lysozyme had no effect, human milk a-lactalbumin had a small, but significant inhibitory effect (at concentrations of added protein > 1 ~Mthere was a 25% inhibition), while human milk lactotransferrin
2001 RELATIVE ACTIVITY
(~)
I
oi o
10
O
2ooI RELATIVE / ACTIVITY [ I~)
i A looT. :
c
0.0
.
02
0.4
0.g
0.8
1.0
1.2
,;c_~-
2,0
[PROTEIN] (pM)
FIG. 1. Variation of the relative initial rate of human milk lipase-catalyzed decomposition of 4-nitrophenylacetate in 0.1 M Tris/HCl buffer containing 1.6% (v/v) acetonitrile at pH 7.5, 25°C, in the presence of (O, O) human milk lactotransferrin, (~, a) human colostrum IgA. Open symbols, in the absence of taurocholate; closed symbols, in the presence (2 mM) of taurocholate.
and human colostrum IgA were strong inhibitors and almost equal in their effect. As little as ~0.2 tzM human lactotransferrin present in the assay mixture caused a 70% inhibition of the HML activity, compared with the activity in the absence of lactotransferrin. This level remains constant as the concentration of lactotransferrin was increased to 0.8 uM but, at 2 tsM, 85% inhibition was observed. Results obtained with horse heart myoglobin, bee venom melittin and chicken egg white lysozyme are presented in Fig. 3 and those for porcine pancreatic lipase in Fig. 4. Under the conditions used, myoglobin (< 10 /zM) increased the esterase activity of HML in the absence of TC, whereas melittin (<12 #M) and chicken egg white lysozyme (<22 ~tM) decreased the activity to ca. 50%. This activity was achieved in the presence of 0.4 #M melittin or chicken lysozyme and at higher concentrations the activity remained almost constant at this depressed level. The activity of HML against PNPA (in the absence of bile salt) was completely inhibited, Journal of Colloid and Interface Science, Vol.
112, N o . 2, A u g u s t 1986
492
O'CONNOR AND WALDE 2ooI
hydrolytic enzymes are able to catalyze the decomposition of ester substrates. Sperm whale metmyoglobin (21), bovine mercaptalb u m i n (15), and h u m a n serum albumin (16, (~) 22) catalyze, for example, the decomposition of PNPA, a c o m p o u n d which is subjected to general acid-general base catalysis in both aqueous (23) and nonaqueous solutions (24). ~oo1 RELATIVE / In the present work we have found that ACTIVITY a m o n g the proteins tested bovine serum al[ ~ ~00[ b u m i n was the most effective in releasing pnitrophenol from the water soluble substrate PNPA. Due to this high activity the effect of 0 i 2 3 L s 6~ a serum albumin on the activity of bile salt[PROTEIN]()~M) stimulated lipase could not be studied. In the FIG. 2. Variation of the relative initial rate of human other cases, no biphasic reaction was observed milk lipase-eatalyzed decomposition of 4-nitrophenyl- and the extent, if any, of the catalysis induced acetate in 0.1 M Tris/HC1 buffer containing 1.6% (v/v) by the presence of added protein seems to be acetonitrile at pH 7.5, 25°C, in the presence of (©, O) human milk a-lactalbumin, (A, A) human milk lysozyme. related to the n u m b e r of imidazole groups (histidine residues) of the protein molecule Open and closed symbolsas in Fig. 1. RELATtVE [ ACTIVITY
~OOT: Ao
o
o
o
o
under the conditions used, by the presence of 1 ~tM pancreatic lipase. Even in the presence of 0.2 u M pancreatic lipase the activity of H M L was ~ 5-10% compared with the H M L activity in the absence of protein. Under the conditions used, neither myoglobin nor melittin had any effect on the activity of H M L in the presence of TC. Porcine pancreatic lipase had a small inhibitory effect (30% in the presence of 1 ~ M protein) compared with the activity in the absence of pancreatic lipase, while chicken egg white lysozyme had a small catalytic effect (130% in the presence of 8 # M protein). Further experiments were performed to show that t h e effect of pancreatic lipase u p o n H M L is reversible; inhibition can be reversed by adding TC after addition of pancreatic lipase to HML. The incubations were carried out both in the absence of CH3CN and substrate (PNPA) and in their presence. DISCUSSION It has previously been reported that several proteins which are not normally classified as Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
COO 13 [3
300 RELATIVE ACTIVITY (%) 2O0
0
0
1001 o
&
a~
,y
2°° I
RELATIVE ACTIVITy
:,
(%) 100~ •
0
•
2
~
L,
I
6
8
•
II•
I0
4/-~-12 22 2t,
[PROTEIN](pM)
FIG. 3. Variation of the relative initial rate of human milk lipase catalyzed decomposition of 4-nitrophenylacetate in 0.1 M Tris/HCl buffercontaining 1.6%(v/v) acetonitrile at pH 7.5, 25°C, in the presenceof(O, g) chicken egg white lysozyme,(A, A) melittin from bee venom, (D, u) horse heart myoglobin.Open and closed symbolsas in Fig. 1.
STUDIES IN BILE SALT SOLUTIONS, XVII
493
200t
was little affected by these steroidal surfactants. In the light of this result, it is not surprising ACTIVfTY (%) that there was an absence of any additional 100 effect, in the presence of TC, in these present o experiments. o _ _ Q r, _ _ ,~ It has repeatedly been reported that proteins "unfold" at the interfaces which separate a polar from an apolar phase (26). The majority 2oo1 RELATIVE ] of the polar groups of native globular proteins ACTIVITY ~ • c~> ; " in aqueous solution are now known to be at the surface of the molecules in contact with the solvent, while most of the hydrophobic groups are buried inside where they ensure in0.2 0.3 0J~ 05 0.6 1.0 1.1 12 0.0 0.1 ternal stability. Therefore the proximity of an [PROTEIN] (pt4) amphipathic compound may be expected to FIG. 4. Variation of the relative initial rate of human alter several groups in the protein and thus milk lipase catalyzed decomposition of 4-nitrophenylmodify its three-dimensional structure. The acetate in 0.1 M Tris/HC1 buffer containing 1.6% (v/v) acetonitrile at pH 7.5, 25°C, in the presence of porcine degree of surface denaturation may depend on pancreatic lipase. Open and closed symbols as in Fig. 1. the hydrophobicity of the amphiphile which is adsorbed, and in the situation when the protein is an enzyme, interfacial adsorption may (and in particular the number of histidine res- induce progressive inactivation. idues lying on the surface of the molecule). The effect on the activity of human milk Thus, bovine serum albumin, with 17 histidine lipase by human lactotransferrin, human coresidues, is strongly catalytic, porcine pan- lostrum IgA, human milk oMactalbumin, bee creatic lipase (10 histidine residues) and myo- venom melittin, horse heart myoglobin, and globin (11 residues) are moderately catalytic, porcine pancreatic lipase can, in each case, be while human lactotransferrin (8 residues), hu- accounted for by postulating sorption of these man milk lysozyme, chicken egg white lyso- proteins on the surface of the enzyme. The zyme, and human milk a-lactalbumin (each magnitude of the inhibition will be related to with 1 residue) and melittin (zero histidine the capacity of the enzyme to interact with the residues) are ineffective. Breslow and Gurd protein. The absence of any effect in the pres(21) have shown that 6 imidazole groups of ence of human milk lysozyme indicates either native metmyoglobin are freely reactive and that there is insignificant affinity o f the enzyme responsible for the catalytic decomposition of for this protein or that, if sorption does occur, PNPA by this protein. Although human lac- it does not have an effect on the rate of cataltotransferrin has 8 histidine residues per mol- ysis. In each of the cases in which inhibition was ecule, under the conditions used no catalytic effect on the hydrolysis of PNPA could be ob- observed in the absence of bile salt there was served. This result indicates that at least some an absolute requirement of bile salt to relieve of these imidazole groups may lie buried be- inhibition of the lipase system by protein. The neath the protein surface or are less reactive, concentration of bile salt used, 2 mM, could not fully relieve the inhibition in the case o f due to local environmental effects. O'Connor and Wallace (25) studied the ef- porcine pancreatic lipase and human milk fect of a range of bile salts on the nonenzy- lactotransferrin. It appears that the bile salt competes with matic hydrolysis of PNPA and other esters and found that the stability of these compounds the enzyme for the protein interface and, in a RELATIVE
ioo~.
I
•
Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
494
O'CONNOR AND WALDE
concentration above its critical micelle con- of bile salt-stimulated lipase is achieved by the centration, it clears the enzyme interface of monomeric bile salt (28). Certainly, in the case protein. A specific reunion of the unmodified of the cholates, activity increases with increasenzyme with the substrate, and a restoration ing bile salt concentration up to the critical of catalytic activity is affected in the presence micelle concentration and then remains at the of bile salt. The restoration of a constant ac- constant level maintained by the equilibrium tivity, even in the presence of excess protein, concentration of bile salt monomer. Such an is an effect of the physical separation of protein effect of bile salt in separating the protein from from the enzyme. A possible reaction scheme the enzyme is a general surfactant effect and is given in Fig. 5. The reversible inactivation supports the conclusion that lipase inactivaof the enzymic activity, noted in the presence tion is not related directly to the change in of pancreatic lipase, supports this scheme interracial tension brought about by the prowhich describes the formation of a bile salt tein but to desorption of lipase from its submicelle-protein complex which does not in- strate. teract with the enzyme. Such a postulate is The activation of the activity of human milk consistent with the overwhelming evidence lipase by horse heart myoglobin and the rethat the modification of pancreatic porcine li- versal of this activation in the presence of bile pase activity by bile salts is linked to a variation salt is not inconsistent with the above discusin the adsorption capacity of the enzyme aris- sion. We have already suggested that nonionic ing from a modification of the interface (27). surfactants interact with the enzyme and We have previously suggested that activation thereby induce a conformational change which
+ S '-
NO ESTERASE ACTIVITY
ESTERASE ACTIVITY
FIG. 5. Tentative scheme for the anchoring of h u m a n milk lipase, L, by protein, P, and for reversal of deactivation in the presence of an equilibrium mixture of bile salt micelles and monomers. S is an ester substrate. The molecules are not drawn to scale. Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
STUDIES IN BILE SALT SOLUTIONS, XVII
leads to an increase in the binding of the substrate to the enzyme (1). In a similar way, we can postulate that the interaction ofmyoglobin with human milk lipase causes a conformational change which is favorable for enzymatic activity, but that the presence of bile salt separates the protein from the enzyme, thereby restoring the original conformation and the activity observed in the absence of protein. It might have been assumed that the magnitude of the interaction between the protein and the enzyme, and thereby the degree of catalysis and inhibition, was related to the hydrophobicity of the protein. A method has been devised for calculating the average hydrophobicity of proteins, H~av, from their amino acid composition, without taking into account possible contributions caused by the secondary structure of the protein (29). A number of alternative hydrophobicity scales has been constructed simply by comparing the ratio (R) of the frequencies of occurrence (~) of whatever particular side chain one wishes to stress. One feature of proteins that is fairly reliable is the tendency of charged or very polar residues to be external. A particular ratio scale (R3) is constructed from
R3-
k
[1]
J
where k represents the polar side chains (Lys + Arg + His + AsX + G1X), j represents apolar side chains (Ile + Tyr + Phe + Leu + Val + Met), and ~pis the frequency of occurrence ofk orj per mole protein (30, 31). This choice of residues is arbitrary, but convenient, since it spreads out different proteins over a relatively wide scale. R3 and H,av show some anticorrelation, but they are by no means purely inverse measures. R3 allows a fairly clear distinction between internal and external membrane proteins, whereas H~av does not. We calculated values of H~avand R3 for the proteins which have been used to modify the
495
activity of HML in this study, but we found no relationship between enzymatic activity and those values. This result gives further support to the conclusion that the activity is not related to the surface tension of the protein. We also compared the activity with the relative values of the isoelectric points of the proteins but found that the activity (or lack of it) did not seem to be directly related to the acidity of the surface of the protein. ACKNOWLEDGMENTS Equipment grants from the Research Committees of the Universities of New Zealand Grants' Committee and the University of Auckland and the award of a University of Auckland Postdoctoral Fellowship (to PW) are gratefully acknowledged. We are indebted to the staff of National Women's Hospital, Auckland, for collecting the breast milk; to Dr. B. E. C. Banks, Dr. P. M. Barling, and Dr. S. V. Rumball for the kind donation of chemicals; to the members of the Biochemistry Department, University of Auckland, for assistance in purification of the enzyme; and to the New Zealand Foundation for the Newborn for financial assistance. REFERENCES 1. O'Connor, C. J., and Walde, P., Langmuir, 2, in press (1986); O'Connor, C. J., Stockley, I. C., and Walde, P., N.Z. Med. J. 98, 306 (1985). 2. Brockerhoff, H., J. Biol. Chem. 246, 5828 (1971). 3. Borgstrrm, B., and Erlanson, C., Gastroenterology 75, 382 (1978). 4. Bl~ickberg, L., Hernell, O., Bengtsson, G., and Olivecrona, T., J. Clin. Invest. 64, 1303 (1979). 5. Gargouri, Y., Julien, R., Sugihara, A., Verger, R., and Sarda, L., Biochim. Biophys. Acta 795, 326 (1984). 6. Macritchie, F., Adv. Protein Chem. 37, 283 (1978). 7. Jensen, A. A., in "Residue Reviews" (F. A. Gunther and J. D. Gunther, Eds.), Vol. 83, pp. 2-128. Springer-Verlag, New York, 1983. 8. Ribadeau-Dumas, B., Endeavour 7, 80 (1983). 9. Habermann, E., Science 177, 314 (1972). 10. Habermann, E., and Jentsch, J., Hoppe-Seyler's Z. Physiol. Chem. 348, 37 0967). I I. Bl~ickberg,L., and Hernell, O., Eur. J. Biochem. 116, 221 (1981). 12. Jolles, J., and Jolles, P., Biochemistry 6, 411 (1967). 13. Desnuelle, P., in "The Enzymes" (P. D. Boyer, Ed.), 3rd ed., Vol. VII, pp. 575-616. Academic Press, New York, 1972. 14. Fasman, G. D., Ed., in "C. R. C. Handbook of BioJournal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
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O'CONNOR AND WALDE
chemistry and Molecular Biology Proteins, 3rd ed.," Vol. II, pp. 383-545, C. R. C. Press, Boca Raton, Fla., 1976. 15. Tildon, J. T., and Ogilvie, J. W., J. Biol. Chem. 247, 1265 (1972). 16. Means, G, E., and Bender, M. L., Biochemistry 14, 4989 (1975). 17. O'Connor, C. J., and Walde, P., N.Z. Med. J. 98, 114 (1985). 18. Blanc, B., WorldRev. Nutr. Diet36, 1 (1981). 19. Bezkoravalny, A., J. Dairy Sci. 60, 1023 (1977). 20. S~m~riva, M., Chapus, C., Bovier-Lapierre, C., and Desnuelle, P., Biochem. Biophys. Res. Commun. 58, 808 (1974). 21. Breslow, E., and Gurd, F. R. N. J. Biol. Chem. 237, 371 (1962). 22. Kurono, Y., Maki, T., Yotsuyanagi, T., and Ikeda, K., Chem. Pharm. Bull. 27, 2781 (1979). 23. O'Connor, C. J., and Wallace, R. G., Aust. J. Chem 37, 2559 (1984).
Journal of Colloid and Interface Science, Vol. 112, No. 2, August 1986
24. O'Connor, C. J., Lomax, T. D., and Ramage, R. E., Adv. Colloid Interface Sci. 20, 21 (1984). 25. O'Connor, C. J., and Wallace, R. G., Aust. J. Chem. 37, 1881 (1984). 26. James, L. K., and Augenstein, L., Adv. Enzymol. 28, 1 (1966). 27. Vandermeers, A., Vanderrneers-Piret, M. C., Rathe, J., and Christophe, J., Biochem. Biophys. Res. Commun. 69, 790 (1976). 28. O'Connor, C. J., and Wallace, R. G., £ Colloid Interface Sci. 102, 539 (1984). 29. Fasman, G. D., Ed., in "C. R. C. Handbook of Biochemistry and Molecular Biology, Proteins, 3rd ed.," Vol. I, pp. 209-210. C. R. C. Press, Boca Raton, Fla., 1976. 30. Cantor, C. R., and Schimmel, P. R., "Biophysical Chemistry," Part I, pp. 55ff. Freeman, San Francisco, 1980. 31. Barrantes, F. J., Biochem. Biophys. Res. Commun. 62, 407 (1975).