The kinetics of the partial digestion of bovine a-lactalbumin (a-LA) by trypsin, a-chymotryp- sin, and pepsin was monitored by lactose synthase activity, HPLC, ...
Journal of Protein Chemistry, Vol. II, No. I, 1992
Proteolytic Digestion of a-Lactalbumin: Physiological Implications Yukihiko Hirai, 1'2 Eugene A. Permyakov, 1'3 and Lawrence J. Berliner 1'4 Received August 16, 1991
The kinetics of the partial digestion of bovine a-lactalbumin (a-LA) by trypsin, a-chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of a-LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)-a-LA > Zn(II), Ca(II)a-LA> apo-a-LA. The HPLC digestion patterns of Ca(II)-a-LA and Zn(II), Ca(II)-a-LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo-a-LA were quite different, reflecting the existence of the thermally induced denaturation states of apo-aLA within this temperature region. Occupation of the first Zn(Ii)-binding site in Ca(II)-loaded a-LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of a-LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(lI))-specific site does not cause any drastic changes in the overall structure of a-LA. The acidic form of a-LA (at pH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or a-chymotrypsin at neutralpH. Complexation of a-LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to a-LA may have physiological significance (eog., for nutritional transport). KEY WORDS: a-Lactalbumin; pepsin; trypsin; a-chymotrypsin; digestion; bis-ANS.
1. INTRODUCTION
gatactosyltransferase displays a low affinity for glucose (Kin ~ 1.0 M), while in the presence of a-LA, the Km is lowered to the millimolar range (Fi~tzgerald et al., 1970). The tertiary structure of a-LA is very similar to that of lysozyme (Smith et aL, 1987). It has been shown that a - L A is a metal ion-binding protein (Hiraoka et al., 1980) with one strong Ca(iI)-binding site [which can also bind Mg(lI), Na(I), and K(I)] as well as separate Cu(II) and Zn(II)-binding sites (Permyakov et al., 1981a, b, 1985, 1987; Murakami and Berliner, 1983; Musci and Berliner, 1985a, b). The binding of Ca(II) to a - L A causes a conformational change, which only slightly alters the main elements of the secondary structure (Dolgikh et al., 1981). On the other hand, the environment around some Trp and Tyr residues changes significantly [e.g., the removal of Ca(II) from a - L A results in an increase in both Trp and Tyr residue accessibility to solvent
Lactose is synthesized in the Golgi apparatus of the lactating mammary cell by the enzyme complex, lactose synthase (E.C. 2.4.1.22), which is composed of an enzyme and a modifier protein. The enzyme, galactosyltransferase, catalyzes the transfer of galactose from UDP-galactose to a 13-(1,4)-linkage to the acceptor, N-acetylglucosamine, or glucose. In the absence of the modifier protein, a-lactalbumin (a-LA), 5 f Department of Chemistry, The Ohio State University, 120 W. 18th Avenue, Columbus, Ohio, 43210. Present address: Department of Biochemistry, Nippon Medical School, Tokyo, Japan. 3 On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR. 4 To whom all correspondence should be addressed. Abbreviations used: a-LA, a-lactalbumin; bis-ANS, 4,4'-bis-[1(phenylamino)-8-naphthalene sulfonate]; EGTA, ethylenediamine tetraacetic acid; HEPES, N-2-hydroxyethylpiperazine-N'-2ethane sulfonic acid.
51 0277-8033/92/0200-005I$06.50/0,~, 1992PlenumPublishingCorporation
52
molecules (Ostrovski et al., 1988; Berliner et aL, 1987 ; Permyakov et al., 1985)]. Apo-a-LA also has increased affinity for the apolar fluorescent probe, bisANS (Musci and Berliner, 1985a). In addition, several other experimental methods have shown that Ca(II) removal shifts a-LA toward an intermediate state called the "molten globule," which possesses intact secondary structure, but lacking intact tertiary structure. The cations Ca(II), Mg(II), Na(I), and K(I) stabilize the protein against thermal denaturation, the effect being the most pronounced in the case of Ca(II) (Hiraoka et al., 1980; Hiraoka and Sugai, 1984; Permyakov et al., 1985). In contrast, the binding of millimolar Zn(II) or Cu(II) to Ca(II)-loaded a-LA shifts the conformation in the opposite direction, transforming it to an "apo-like" conformation, decreasing thermostability and inducing protein aggregation (Murakami and Berliner, 1983; Musci and Berliner, 1985a, b; Permyakov et al., 1988a, b). Remarkably, the physiological significance of these metal ion-binding phenomena are still not yet entirely understood. A noteworthy feature of a - L A structure is the acidic conformational transition occurring between pH 3 to 4, which is accounted for, in part, by a competition between Ca(lI) and protons for the carboxyl side chains comprising the calcium binding site (Permyakov et al., 1981b, 1985). At highly acidic pH (
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time (min) Fig. 4. Time course of the digestion of 63/~M a-LA by 38 pM trypsin (or 38 pM pepsin) at pH 7.7 (or pH 2.2) as monitored by difference UV absorbance at 295 rim. The curves are normalized to the maximum amplitude. Curve 1: tryptic digestion of Ca(II)-aLA (8 mM CaC12, 10 mM HEPES, pH 7.7); curve 2: tryptic digestion of apo-a-LA (1.8 mM EGTA, 10 mM HEPES, pH 7.7); curve 3: tryptic digestion of Cu(II), Ca(II)-a-LA; in this particular case, the a-LA concentration was 6.8 p M in order to avoid aggregation/ precipitation caused by high concentrations of Cu(II) (1.9 mM CaCI2, 9.6 pM CuCI2, 3.8 pM trypsin, 10 mM HEPES, pH 7.7) ; curve 4: peptic digestion of a-LA (10 mM sodium acetate, pH 2.2).
attack (Permyakov et al., 1991)]. However, it is important to note that the digestion patterns for both C a ( I I ) - a - L A and Zn(II), C a ( I I ) - a - L A were quite similar (Fig. 2, Table II). This suggests that saturation of the first Zn(II) binding site in C a ( I I ) - a - L A does not induce major structural changes, which is also consistent with recent spectroscopic studies on Zn(ll) binding to a - L A (Permyakov et al., 1991). Qualitatively similar results were obtained for chymotryptic digestion of these various forms of a - L A (Tables I and II). Figure 3 depicts the loss of a - L A activity during pepsin digestion (curve I, 0.07 tiM pepsin, p H 2.2), and, simultaneously the difference absorption at 295 nm ( c u r v e 2). There was generally good agreement between the two methods. Although the two curves do not completely coincide, we note that they reflect both different aspects of the digestion process and different regions of the a - L A structure. Figure 4 depicts the difference absorption time course of several a - L A metal-bound states in the presence of trypsin ( p H 7.7) and pepsin ( p H 2.2), respectively. Here the pepsin concentration was much higher (38 p M ) than in Fig. 3 (0.07/IM), which accounts for the much faster rate. Peptic digestion of the acid form of a - L A was quite rapid, as was that for tryptic
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time(min)
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time (min) Fig. 5. Time course of a-LA (lactose synthase) activity during the digestion of 63 #M a-LA by 0.07 #M pepsin: (A) in the presence of 163/IM bis-ANS (10 mM sodium acetate, pH 2.2, 37°C). Each experimental point represents the average of three measurements. (B) Time course of the digestion of 64 pM a-LA by 38 pM pepsin in the absence (curve 1) and presence (cur~e 2) of 65 ,uM bis-ANS, as monitored by difference UV absorbance at 295 nm. All other conditions were identical to those in Fig. 4.
digestion of the apo-protein. This result is in line with previous conclusions that the apo- and acid forms of a - L A are very similar (Permyakov et al., 1985). Thus, it seems straightforward that when free aLA is absorbed into the stomach, it is rapidly digested by pepsin. On the other hand, if the acid form were
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56
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Fig. 7. Time course of the peptic digestion of 71 pM lysozyme at pH 2.2 (29 pM pepsin, 10 mM sodium acetate, 37°C) in the absence (curve 1) and presence (curve 2) of 75 pM bis-ANS as monitored by difference UV absorbance at 295 nm.
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time (min) Fig. 6. Time course of the tryptic digestion of 64 pM a-LA (38 pM trypsin, 10mM HEPES, pH 7, 37°C) in the absence (curves A, B, C) and presence (curves 2A, B, C) of 65 pM bis-ANS as monitored by difference UV absorbance at 295 rim: A, Ca(II)-aLA (1.8 mM CaCI2); B, apo-a-LA (1.8 mM EGTA); C, Cu(ll), Ca(II)-a-LA (3.8pM trypsin, 6.8pM a-LA, 1.9rnM CaCI2, 9.6 pM CuCl2).
able to bind low molecular weight apolar substances, this might reduce the peptic digestion rate. As a model system, we chose the fluorescent dye bis-ANS. Fluorescence titrations of bis-ANS with the acid form aLA at pH 2.2 yielded an apparent dissociation constant of 5.9pM (data not shown), which was in excellent agreement with the values obtained for the apo-protein at pH 3.9-7.4 by Musci and Berliner (1985a), confirming also that the acid state of a-LA also has high affinity for apolar substances. We then monitored a-LA activity during (0.07 pM) peptic digestion in the presence of bis-ANS (Fig. 5A), where
the difference UV showed little or no change (data not shown). At much higher pepsin concentration (38pM), where the rate of digestion is significant (Fig. 5B, curve 1), the UV difference absorption also increased (Fig. 5B, curve 2). The reversal in direction of these pepsin effects in the absence and presence of bis-ANS may reflect different pathways of protein cleavage. Figure 6 depicts the change in difference absorbance for tryptic digestion ofCa(II)-, apo-, and Cu(lI), Ca(II)-a-LA in the absence and presence of bis-ANS. Since Cu(II) and Zn(II) compete for the same sites on a-LA, inducing similar structural changes, (Musci and Berliner, 1985a, b; Permyakov et al., 1987) and since Cu(II) causes much less pronounced aggregation problems compared to Zn(II), the Cu(II)-bound states were chosen for the spectroscopic measurements. Again, here, binding of bis-ANS to a-LA increases the resistance to digestion. This "protective effect" was more pronounced with apo-a-LA, which also has the highest affinity for bis-ANS (Musci and Berliner, 1985a). In order to rule out any contributions due to bis-ANS inhibition of pepsin or trypsin, since both pepsin and trypsin can bind bis-ANS, we monitored the digestion of lysozyme.6 As shown in Fig. 7, the presence of bis-ANS both reversed the direction of the pepsin effect and altered the digestion pathway (since at least two digestion steps are apparent in curve 2), but did not protect lysozyme against 6Bis-ANS dissociation constants: for pepsin Kd=7.3pM at pH 2.2; for lysozyme Kd=8.2+ 1.5 ,uM (this work).
Kinetics of a-Lactaibumin Digestion
digestion. Therefore, the protective effect of bis-ANS is specific for a-LA, and does not involve pepsin inhibition.
4. CONCLUSIONS • From these results it is clear that tryptic or chymotryptic digestion of Ca(II)-a-LA in the pancreas or intestines would be extremely slow were it not for the partial digestion which first occurs in the highly acidic environment of the stomach where Ca(II) is released from a-LA and peptic cleavage occurs. The binding of low molecular weight apolar molecules in milk might also protect a-LA against digestion in the stomach, thus allowing a-LA to serve as a transport protein for these substances to pass the stomach to the intestines where they might be utilized. Preliminary studies on the characterization of apolar constituents in milk which might bind to a-LA are in progress. For example, intrinsic fluorescence studies show that folic acid binds to a-LA at both pH 2,0 and 8.0, although this ligand does not appear to protect a-LA against peptic digestion (E.A. Permyakov, unpublished results).
ACKNOWLEDGMENTS
The authors thank Dr. Dore C. Meinholtz for helpful comments. This work was supported in part by a grant from the USPHS (GM 40778).
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