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Interaction between the basic inhibitor of bovine pancreas and chymotrypsin and trypsin. Selective anthraniloylation of the maleylated inhibitor
J. Mol. Biol.
(1974) 84, 523-538
Interaction Between the Basic Inhibitor of Bovine Pancreas and Chymotrypsin and Trypsin. Selective Anthraniloylation of the Maleylated Inhibitor YERUDIT
ELKANA
Department of Biological Chemistry The Hebrew University, Jeru,nalem, Irsael (Received 20 September 1973) The interaction between the maleylated basic pancreatic inhibitor, anthraniloylated on its lysine-15 residue, and chymotrypsin is studied by fluorescence intensity, fluorescence polarization, circular dichroism, circular polarization of fluorescence and sedimentation. These measurements show that the interaction takes place through the entrance of the anthraniloyl group into an asymmetric environment in which it is rigidly held. The dissociation constant of the complex is 2.5 x lo-* M. The interaction between the modified inhibitor and trypsin takes place through a site which is not the anthraniloylated lgsine-15 side-chain, yet not far from it.
1. Introduction The interaction between trypsin and the basic pancreatic trypsin inhibitor takes place at the inhibitor residue lysine-15 (Chauvet & Acher, 1967; Kress $ Laskowski, 1968). The site of interaction with chymotrypsin on the intact inhibitor has not been unambiguously proved. Kraut & Bhargava (1967) showed that a complex of trypsin and BPT inhibitort does not inhibit chymotrypsin, whereas the inhibitor itself does, thus suggesting that the site of interaction is either the same as the one for trypsin or is covered by the trypsin area that interacts wit,h the inhibitor. In limited proteolysis experiments, Rigbi (1971) has shown that in the partially reduced inhibitor, lysine-15 and perhaps arginine-39 are loci of attack by chymotrypsin, suggesting that either only the former site or both are the active sites of the native inhibitor toward chymotrypsin. Blow et al. (1972) have shown by model building that the only mode of binding of the inhibitor to chymotrypsin is through the entrance of the side-chain of the inhibitor lysine-15 residue into the specificity pocket of chymotrypsin. In order to get direct proof the role of lysine-15 in the interaction with chymotrypsin, the inhibitor can be labeled at this site with a label which will be sensitive to an interaction with the “tosyl hole” (Sigler et al., 1968) of chymotrypsin. As the inhibitor has no tryptophan residues (Green & Work, 1953), a convenient label is a fluorescent compound capable of accepting energy from the tryptophan residues of chymotrypsin. Haugland & Stryer (1967) labeled chymotrypsin with an anthraniloyl group and found that in the labeled enzyme energy was transferred from the tryptophans to the anthraniloyl group. Although there is no direct Abbreviations used: BPT inhibitor, antbraniloyl (Lys-15 free) maleyleted
basic trypsin BPT inhibitor.
,523
inhibitor
of bovine
pancreas;
AM inhibitor,
524
Y.
ELKANA
evidence that the anthraniloyl is bound to the active site serine-195 of chymotrypsin, it seems likely that the aromatic group is situated in the tosyl hole. Moreover, it was shown by Schlessinger & Steinberg (1972) that bot’h circular dichroism and circular polarization of fluorescence are induced in anthraniloyl-chymotrypsin. In the present papel’ ‘we report studies of the interaction of chymotrypsin and trypsin with the anthraniloyl derivative of maleylated BPT inhibitor. We show that this interaction between the two macromolecules takes place by the entrance of the anthraniloyl group into the tosyl hole of chymotrypsin. Although the modified inhibitor inhibits trypsin, no such interaction between the anthraniloyl group and the active
site of trypsin
can be detected.
2. Materials and Methods N-tosyl-L-phenylalanine chloromethyl-trypsin (lot TRTPCKSIC), a-chymotrypsin (lot CDISLK) and pancreatic trypsin inhibitor compound (lot PICIJB) were purchased from Worthington Bioch&mical Corporation, Freehold, New Jersey. N-tosyl-L-arginine methyl ester was a Miles-Yeda, Rehovot, product. Carbobenzoxy-L-phenylalanine /3-naphthyl ester was prepared by E. Haaa (Haas et aE., 1971). p-Nitrophenyl anthranilate was a gift from A. Carmel (Carmel, unpublished results). Amberlyst A21 was purchased from Serva Feinbiochemica, Heidelberg. N-tosyl-L-lysine chloromethyl ketone-chymotrypsin was prepared according to Shaw et al. (1966). Anthraniloyl-chymotrypsin was prepared according to Haugland & Stryer (1967). The complex of trypsin and BPTinhibitor was purified by passage on Sephadex G75 under the conditions described in the maleylation experiment. Anthranilic acid was recrystallized from water, N-hydroxysuccinimide from ethyl acetate, urea from ethanol and sodium deoxycholate from methanol-ether. Butylamine and dimethylsulfoxide were distilled before use. Maleic anhydride was sublimed. All other mat.erials were of analytical grade. Glass-distilled deionized water was used throughout. Anthranilic acid N-hydroxysucoinimide ester was prepared according to the procedure of Anderson et a.!. (1904). The product was recrystallized 3 times from ethanol. Yield, 86%; m.p., 163’C. Analysis calculated for C11H,004Na: C, 56.41; H, 4.27; N, 11.97. Found: C, 56.03; H, 4.66; N, 12.28. Fluorescence Absorption spectra were taken on a Gary 14 recording speotrophotometer. intensity was measured on an Aminco-Bowman spectrophotofluorometer with J&novia 160 W Xenon or 200 W Xenon-Mercury lamps and an RCAlP28 photomultiplier. Fluorescence polarization measurements were made on the same instrument equipped with Glan prisms supplied by Aminco. Polarization of fluorescence is defined as 2, = (P,, - Pl)/ (E;, + p,), where E;, and FL are the intensities of the light emitted parallel and perpendicular to the direction of polarization of the exciting light. If the polarization is measured in a spectrofluorometer one has to take into account polaxization artifacts introduced by the grating of the emission monochromator. This effect was corrected for according to the method of Azumi & McGlynn (1962) by measuring the grating correction factor, a. Introducing this factor G, p becomes p = (PII - GFJ/(F,, + GPI). F,, and FL were each corrected for the appropriate blank readings. Fluorescence polarization was excited at 313 nm. Temperatures were regulated with a constant temperature accessory. Fluorescence intensity measurements were performed at 25”C, on solutions whose optical density at the wavelength of excitation was below 0.1. Circular di&roism was measured on a Cary 60 spectropolarimeter equipped with a 6002 dichroism aooessory. Measurements of circular polarization of fluorescence were made on an instrument built and described by Steinberg & Gafni (1972). Polyaorylamide gel electrophoreaw were run on a Yeda (Rehovot, Israel) instrument according to Davis (1964) on 11% gels stained with Coomasie Brilliant Blue R (Weber BEOsborn, 1969). Ultrafiltration was carried out with Amicon cells and Di&o membranes. A Beckman model E analytical ultracentrifuge was used for sedimentation studies. Protein hydrolyses were carried out in 6 N-HCl in evacuated sealed tubes for 20 h at
MODIFIED
INHIBITOR-CHYMOTRYPSIN
COMPLEX
525
llO”C, and amino acid analyses were performed on a Beckman amino acid analyzer model 120C. Complex formation between AM inhibitor and chymotrypsin was followed by the increase in fluorescence intensity at 415 run and wss considered complete when the intensity became constant on further addition of chymotrypsin. Peptide mapping of AM inhibitor was carried out on the tryptic hydrolysis of performic acid oxidized AM inhibitor according to Chauvet et al. (1906). The peptides were identified by amino acid analysis. Quantitative compositions could not be obtained owing to the small quantity of peptide in hand and the errors of analysis. The concentration of AM inhibitor and its extinction coefficient in the anthraniloyl absorption band were determined from the amino acid analysis and the absorption spectrum. (a) Anthraniloyl butylamide Butylamine (26 ml, 22 mmol) was added drop-wise to a stirred solution of anthcanilic acid N-hydroxysuccinimide ester (1 g, 4.3 mmol) in ethyl acetate (50 ml). A white precipitate appeared, which was filtered off after stirring the reaction mixture overnight at room temperature. The filtrate wss extracted with a 5% solution of sodium bicarbonate to remove N-hydroxysuccinimide, rinsed 3 times with water and dried over anhydrous sodium sulfate. After evaporation the yield was 0.5 g (60%). The product is amorphous. Analysis calculated for C,,H,,ONz: C, 6875; H, 8.33; N, 1.4.58. Found: C, 68.60; H, 8.20; N, 14.81. (b) MaleyktedfLys-15 free) BPT inhibitor Maleylation of the BPT inhibitor-trypsin complex was carried out according to the procedure of Fritz et aE. ( 1969), with the following modifications : the concentration of the complex was 10 mg/ml ; to a total of 216 mg, 1.5 g of maleic anhydride were added; the final concentration of urea was 6 M and that of ammonium sulfate 25% saturation. The reaction mixture was directly loaded on a Sephadex G75 column (140 cm x 4 cm) and eluted with 0.02 M-ammonium acetate. The yield of maleylated BPT inhibitor was 90%. The calculated degree of maleylation (Butler et al., 1969) was 4.7 -& 0.1 (see Results). (c) Anthranikyl (&/8iTE-15) maleykxted BPT inhibitor (AM inhibitor) 50 mg (210 pmol) of anthranilic acid N-hydroxysuccinimide ester in 5 ml dimethylsulfoxide were added to 40 mg (5-7 ~01) of maleylated (Lys-15 free) BPT inhibitor in 5 ml of 0-l M-triethanolamine buffer, pH 8.0, and the reaction mixture was allowed to stand at room temperature for 48 h. Removal of unreacted and hydrolyzed reagent and solvent wss achieved either by gel filtration through Sephadex G25 or by ultrafiltration through a Diaflo UM2 membrane with 0.02 M-ammonium acetate. In order to separate the desired product from unreacted maleylated BPT inhibitor as well as from traces of BPT inhibitor, Amberlyst A21 (Oeterman, 1971) was used. Preliminary experiments showed that BPT inhibitor was not adsorbed on the resin and that maleylated BPT inhibitor wss eluted at 0.07 N-NaCl and 10 to 15% ethanol. Elution of the desired AM inhibitor from the Amberlyst A21 column (5.5 cm x 1.9 cm) wss carried out by the following steps: loading the desalted reaction mixture and rinsing with 10s3 M-TriseHCl buffer, pH 8.0, containing 0.01 N-NaCl; rinsing with the same buffer containing 1.5 N-NaCl; re-equilibrating with the initial buffer; rinsing with the initial buffer containing 30:/o ethanol. In all these steps elution was continued until no more absorbing materisl could be detected in the eluant. Finally a linear gradient (of a 20-fold vol. excess over the bed resin) of 10V3 M-TriseHCl buffer, pH 8.0, 30% ethanol and 0.01 N to 0.3 N-NaCl was applied. Assay was made by reading the absorbance at 280 nm and at 310 nm snd the fluorescence intensity at 415 nm, excited at 313 run. AM inhibitor was eluted at 0.15 N-Nail, and wss identified by its absorption spectrum (see Results). The fractions containing AM inhibitor were pooled (67% yield), desalted either on Sephadex G25 after reducing the volume by evaporation or, alternatively, by ultrafiltration through a Diaflo UM2 membrane with 0.02 M-ammonium acetate, and lyophilized. The final yield of AM inhibitor wss 50%.
626
Y. ELKANA
(d) Determination of inhibition constant.9 (KI) Inhibition constants (Ki) were determined from Dixon plots, which are obtained by plotting the reciprocal of the initial enzymatic reaction velocity as a function of inhibitor concentration using substrate concentration as a parameter. Reaction mixtures were prepared by incubating the enzyme with the inhibitor at the required concentration for 5 min at 25°C. Reactions were started by adding the substrate to the incubation mixture. Chymotrypsin (final concn 0.1 pg/ml) was assayed with carbobenzoxy-r.-phenylalanine @aphthyl ester as substrate by recording the increase in fluorescence intensity at 420 nm (Haas et al., 1971). The solution contained buffer, 20% dimethylformamide and 1.6% sodium deoxycholate. Trypsin (final concn 0.7 pg/ml) was assayed with tosyl-L-arginine methyl ester by recording the absorbance at 246 nm (Hummel, 1959) on a Gilford model 2000 recording spectrophotometer. As velocities were constant for several minutes, the system was considered to be in a steady state and tho usual inhibition equations are therefore valid. (e) Determination of interaction wnatante (K) Interaction constants between AM inhibitor and chymotrypsin were determined by measuring the increase in the fluorescence of the anthraniloyl moiety of the inhibitor upon addition of chymotrypsin. The measurement was carried out by titrating the inhibitor in the cuvette with increasing amounts of ohymotrypsin. The fluorescence intensity was measured by exciting both at 303 nm and at 313 nm, at a band width of k 5 run, and corrected for readings obtained with the enzyme alone at the same concentrations. The excitation wavelengths were chosen so that at 303 nm tryptophan fluorescence of the enzyme was excited. At 313 nm the only species excited is the anthraniloyl moiety of the inhibitor. The fluorescence intensity (F) is a sum of two contributions, that of the free inhibitor (I) and that of the complexed inhibitor (EI) :
F = 411 + B[EIl, where CLand /3 are constants independent inn the relations
of the concentrations
[I] = [I],, - [EI] ([El is the concentration F = a[Ik, + -$[Elo ‘-” The subscript Differentiating
(1) of the components.
Insert-
[II [El
and K = [EIl
of free enzyme) into equation
+ mo + K-d/(([Elo
(1) we obtain:
+ VI0 + w
- 4~w0m0~1.
0 denotes total concentrations. and taking the limit at [El, -+ 0 we get
The quantity (/I- a) [Ilo can be obtained from the graph of F vemua [El, by subtracting the initial value from the final value at the plateau. The interaction constant K can thus be obtained directly from the graph. (f) Determination of the e$?&ncy of energy transfer Let us designate the light intensity at the wavelength r\ as L(h), the fluorescence quantum yield as Qr, the yield of energy transfer as & and the extinction coefficients of AM inhibitor and chymotrypsin as eAMi(A) and C&A), respectively. The two constants aandjlwillbegivenby: a = L(~Mu&) (4) and
B = uwPxlIwdI(~) Obvhdy
at h = 313 nm, scT = 0.
+ ~dv~tl.
(5)
MODIFIED In reality
INHIBITOR-CHYMOTRYPSIN
the exciting
light
is not monochromatic,
COMPLEX and equations
(4) and (5) change into (4’)
a = #:sQ A)u&)d~
and
B = 4iVU~)~mhWX
527
+ 4,SWV4Nd~l.
The value j3ia is taken from the AM inhibitor titration curve by chymotrypsin given by the ratio of the fluorescence values at the plateau to the initial value. equations (4) and (5) we get for A = 313 run,
(5’) and is From
_s c,"' $'f, 0 =-=
(6)
(3,,,= d’r(l+ g A)= (gJ1 + g A).
(7)
a
313
4:
and for 303 urn
For non-monochromatic
light,
equations
3.
(4’) and (5’) have
to be used.
Results
(a) Ckmacterization of anthraniloyl
(Lys-15) mdeylated inhibitor
AM inhibitor was eluted as the only peak on rechromatography on Amberlyst A21 with a salt gradient in 30% ethanol containing lop3 M-Tris+HCl buffer, pH 8.0, at 0.15 N-NC&~ (Fig. 1). The rechromatographed AM inhibitor, which was desalted and lyophilized, gave one main fluorescent band, one weak band, and several very weak bands on polyacrylamide gel electrophoresis (Plate I). The weak bands can be attributed to inhibitor species that were maleylated to a different extent on their hydroxyamino acid residues (Habeeb & Atassi, 1970), but were too weak to be visible as fluorescent bands.
Fio. 1. Rechromatography of AM inhibitor on Amberlyst AZ1 in 10e3 M-Triu.HCl [N&l]; --o---O-, pH 8.0, 30% ethanol and a salt gradient as indicated. -O--e--, F;;;. --C---o--, A,,,; --a-A----,
buffer, Azso;
528
Y.
ELKANA
The absorption spectrum of (0.75-&0.05) x lob4 M-m inhibitor is given in Figure 2. It shows absorption typical of the anthraniloyl moiety in the wavelength region above 300 nm and typical of the maleylated inhibitor in the region below 300 nm. The extinction coefficient of AM inhibitor at 305 nm is 2950&200 cm2 mmol-I, which is equal to that of anthraniloyl butylamide. For comparison, the spectrum of equimolar concentrations of maleylated BPT inhibitor and anthraniloyl butylamide, at (OG35f0.15) x 10M4 M, is given in the same Figure. The spectra are identical. The fluorescence spectrum of AM inhibitor is given in Figure 8 and is identical to that of anthraniloyl butylamide. From the peptide map of AM inhibitor (Fig. 3) and the list of the expected peptides (Table l), we see that trypsin cleaved only after the endo arginine residues. The only fluorescent spot, that of peptide (1+2), gave no ninhydrin colour, in accordance with a blocked N-terminal and a fluorescent group on lysine-15. Peptide (7+8), which contains the only serine residue of AM inhibitor, appeared twice, probably owing to partial maleylation of the serine residue (Habeeb BEAtassi, 1970), and the resulting greater mobility in a positive direction. Two very weak ninhydrin positive spots could be detected, but did not reveal any peptides by amino acid analysis. These results are consistent with the multiple bands appearing in gel
Wavelength (nml
FIG. 2. Absorption spectra of 0.76 x IO- 4 M-AM inhibitor in 0.1 ~-ammonium acetate (----L of 0.86 x lo- * m-anthraniloyl butylamide (* . * . * . * . e), and of 0,86x lo- 4 x-anthreniloyl butylemide + 0.85 x lo-’ M-meleylated BPT inhibitor (- . - . ) in 0.1 iw-phosphate buffer, pH 7.5.
sr T c
MODIFIED
INHIBITOR-CHYMOTRYPSIN
529
COMPLEX
TABLE 1 Expected tryptic peptides of oxidized AM inhibitor Sequence
Peptides?
1 A g-Pro-Asp-Phe-CysO,H-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro17 16 CysOsH-Lys-Ala-Arg 18 20 Ile-Ile-Arg 21 Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-CysO,H-Glu-Thr39 Phe-Val-Tyr-Gly-Gly-CysOsH-Arg 40 42 Ala-Lys-Arg 43 Asn-Am-Phe-Lys-Ser-Ala-Glu-Asp-CysOsH-MetOssArg 54 68 Thr-CysOsH-Gly-Gly-Ale
I+2
3 4+5
6 7+8 9
t Numbered
according
to Chsuvet
53
et al. (1960).
electrophoresis, and the somewhat high calculated degree of maleylation, 4.7, for one N-terminal and three c-amino groups. The circular dichroism spectra of BPT and AM inhibitors are identical. ;lu’o gross conformational change has taken place in the inhibitor upon modification. AM inhibitor ,+hows no optical activity in the anthraniloyl absorption band.
7.
I-
i
7t0 ,_----> ‘.___A
,/---> ‘./’
1
4t5 =E
,_----. : *-_-_s,
23
6
Fm. 3. Peptide mapping of the tryptic digest of performic acid oxidized AM inhibitor. Peptides are numbered according to Chauvet et al. (1906). Solid outline, ninhydrin positive; dashed outline, weak ninhydrin positive ; hatched area, ninhytlrin negative, fluorescent.
Y.
530
(b) Interaction
of AM
ELKANA
inhibitor
with chynwtrypsin
and trypsin
Figure 4 shows Dixon plots of the inhibition of u-chymotrypsin and trypsin. The K, values of the competitive inhibitions are (3fl) x 10d7 M and (l-3&0.2) x 10V7 M, respectively, for chymotrypsin and trypsin. (i) Fluorivnetric
study of the interaction
between chymotrypsin.
and AM
inhibitor
Typical titration curves of AM inhibitor with chymotrypsin are shown in Figure 5. Both were measured at identical concentrations of AM inhibitor but excited at different wavelengths. We see that there is a rise in fluorescence intensity of the anthraniloyl moiety of AM inhibitor when excited both at its absorption band and at the aromatic absorpt’ion band of chymotrypsin. This rise eventually reaches a plateau, presumably when all the AM inhibitor is bound to the chymotrypsin. The rise in intensity is almost sevenfold when t,he fluorescence is excited in the chymotrypsin tryptophan band and half this value when excited in the anthraniloyl absorption band. There is no shift in wavelength of maximum intensity. From t,he titration curves of the fluorescence intensity as a function of enzyme concentration and by using equation (3), we get K = (2&l) x 1O-8 M. By using equations (6) and (7) and the data of Figure 5 we obtain an energy transfer efficiency of (60-&5)% from the tryptophans of chymotrypsin to t’he ant,hraniloyl group of AM inhibitor upon formation of the complex. No change in anthraniloyl fluorescence intensity is observed when trypsin is added to AM inhibitor (Fig. 5). (ii) Polarization
studies on the formation
of the AM
inhibitor-chymotrypsin
Figure 6 shows the rise in emission anisotropy of AM inhibitor chymotrypsin concentration. Emission anisotropy, r, is defined
complex a function of as (Pii - F,)/
as
T-
-3
-2
-I
0
I
2
3
. -3
-2
-I
0
I
2
3
[AM inhibitor1 (M x 107) (a)
(b)
4. Dixon plots for the inhibition Substrate concentrations were : Fro.
-o-o---, --A--A-, -v--v-,
&ails of assay we in Met&& d in fluorescence arbitrary
by AM inhibitor
of trypsin
(a) and of chymotrypsin
1.1 x 10-4 M; -*--a--, 0.3 x 10-d M; 2-7x lo-* M; --A-A--, 0.8x 10-4 rd; 6.7 x lo- 4 ?a; ---‘I-v---, 1 x10-4 M. and Methods. v is expressed in units of dRacB nm min-1 units min-’ for chymotrypsin.
(b).
for trypsin
MODIFIED
0 Li 0
INHIBITOR-CHYMOTRYPSIN
A-.. LI_II_LuI.-.L I
2
3
4
COMPLEX
531
I I I I ! 1..LLLLL
5
6
[El,
7
8
9
IO
II
(M x10-‘)
Fra. 6. Fluorescence intensity of 2.35 x 10e7 M-Ah% inhibitor as a function of chymotrypsin A) and trypsin (0, n ) concentrations, in 0.04 M-Tris.HCl buffer, pH 8.0. ( l , (1) Excited at 303 nm; (A, A) excited at 313 nm, and normalized at [El, = 0. The curves are calculated according to equation (2) with K = 2.5 x 10e8 M. (0,
(F, + 2F,) (Jablonski, 1960). We see that the emission anisotropy rises as a function of [El,, event)ually reaching a constant level. The functional dependence of T on [El,, is different from that of P on [El,. The anisotropy of a system that is composed of several components is an additive function of the individual anisotropies, ri (Jablonski, 1960) : i
where fi is the fraction of the fluorescence intensity ponent. Equation (8) will take the form :
for the system AM inhibitor-chymotrypsin.
contributed
by the ith com-
The superscripts I and EI denote free
FIG. 8. Emission anisotropy of 2.36 x lo-’ M-AM inhibitor a~ a function of chymotrypsin concentration in 0.04 iv-Tris *HCl buffer, pH 8.0, at 16°C. The curve is calculated a.ocording to equation (X0) with K = 2.6 x 10e8 M.
532
P. ELKANA
and bound inhibitor, respectively. and R, equation (9) becomes
By expressing [I 1 and [El] in terms of [l IS. i E j.
TE’$‘f fvlo
+
?-=
[I]
2
0+
24’f -
?-I
Wlo
1 Plo
+ [Ilo -t K - 2/Wlo + [Ilo + K - &~:I,
f- lllo +- W + PI, + W
- 4l3l,[ll,)l - 4CEloUlo)J (10)
Figure 7 shows the dependence of the degree of polarization of AM inhibitor and of AM inhibitor-chymotrypsin complex on T/T (T, the absolute temperature and 7, the viscosity of the solution). The measurements were carried out isot,hermally at, 20°C on solutions of varying sucrose concentrations. The complex gives a straight line with an intercept at l/p = 3.6 and a slope of 1.3 x 10m4. The polarization values of AM inhibitor are very low, and the experimental error in their measurement is large. A. least squares calculation gives an intercept of 8.2 and a slope of 6*9 x 10m4 for the AM inhibitor polarization. The Perrin-Weber formula for the degree of polarization in the case of a transition moment rigidly held to the molerule is
(11) (Perrin, 1929; Weber, 1952). p, is the fundamental polarization, 7 the decay time of the fluorescence, k the Boltzmann constant and v the molecular volume. If we use this relation, the measured intercepts and slopes of Figure 7 and the relation
we get @I -UI = 7-5.
FIG. 7. Perrin plot of AM inhibitor (0) and of the complex AM inhibitor-chymotrypsin (A) (both at 2.35 IO-’ rd) in aqueous solutions rtt 20°C. Viscosities were taken from the Handbook of Chemistry and Physics, 44th edn.
x
sucr088
MODIFIED
INHlBITOR-CHYMOTRYPSIN
COMPLEX
5 3 :I
(iii) Circular dichroism and circular polarization of jluorescence of the AM inhibitoru-ch ymot ypsin complex The complex between AM inhibit,or and cc-chymotrypsin exhibits induced asymmetry both in absorption and in emission. A positive band appears in the circular dichroism spectrum of the complex in the region of the anthraniloyl absorption. There is also a circularly polarized component in the emission band of the complex. These effect,s are expressed as anisotropy fact’ors (not to he confused with Jablonski’s emission. anisotropy) : for absorption eL-.sR g, = -=E
de F
and for emission gl?=
FL--FP, F/2
AF = F/2’
The subscripts L and R denote left and right-hand circularly polarized components, respectively (Gafni & Steinberg, 1972; Schlessinger & Steinberg, 1972). In Figure 8 1)oth the absorption and the emission anisotropy factors are shown with the absorpt,ion spectrum of anthraniloyl butylamide and the corrected fluorescence and circular dichroism spectra of the complex. We see that the circular dichroism band has a maximum of 325 nm and is shifted by 20 nm to the red relative to the absorption spectrum of anthraniloyl butylamide. The absorption anisotropy band is constant over most of the emission band. Both factors are positive and of similar magnitudes. The complex of AM inhibitor and hrypsin shows no circular dichroism in the ant,hraniloyl absorption band.
FIG. 8. Spectroscopic data for AM inhibitor-cc-chymotrypsin complex. ), Anthreniloyl butylamide absorption spectrum in 0.1 M-phosphate buffer, pH 7.6; AM inhibitor and AM inhibitor-chymotrypsin complex fluoresoence spectra in 0.04 buffer, pH 8.0; -*-.-, circular dichroism of complex expressed 88 de = eL - cR AE in 0.04 :M-Tris.HCl buffer, pH 8.0; -m-e--, absorption anistropy factor, ga = -, of aomplex ; E AF .-, ‘\,--.. :‘, --, emission anisotropy factor, qe - -, of complex. F/2
( --___ (--------), Ma-Tris*HCl
Y.
534
ELKANA
(iv) Interaction of AM inhibitor with anthraniloyl chymotrypsin When AM inhibitor is titrated with anthraniloyl chymotrypsin in the same manner as wit(h chymotrypsin, the rise in fluorescence is proportional to the concentration of anthraniloyl chymotrypsin. From this type of experiment it seems thab no inberaction takes place between the modified inhibitor and the modified enzyme. (v) Sedimentation experiments Table 2 gives the sZo values of AM inhibitor, of a mixture of trypsin and chymotrypsin and of t,he ternary mixture of AM inhibit,or, chymotrypsin and trypsin. In the last experiment (shown in Plat’e II) t,he amount of AM inhibitor exceeded the maximum amount which could have bound to bot’h trypsin and chymotrypsin. In that run, the peak separated into t,wo, one sedimenting with (3.0&0*1) S and the other with 1.4 S. From the ratio between t,he areas of the separated peaks we calculate that the amount of free AM inhibitor is 237” of the total amount of the material in this run. The fraction of total AM inhibitor, bound and free, in this mixture is TABLE 2 Sedimentation
coeficients of AM khibitor, and of their ternary
Run
oj’ trypsin
Composition (in 1 ml) AM inhibitor 2.6 mg trypsin 2 mg chymotrypsin 2.4 mg AM inhibitor 2.6 mg trypsin 2 mg chymotrypsin
Experiments were run in 0.04 BI Tris.HCl buffer, in 0.02 rd.ammonium aoetete, at 59,780 revs/min.
and chyrrwt,:ypsitL,
mixture G20(IkO.1) (S) 1.6 2.9
1.4 and
3.9
pH 8.0, except for AM inhibitor
whioh was
calculated from comparison of the Schlieren patterns at the beginnings of run numbers 2 and 3, where equal amounts of chymotrypsin and trypsin are taken, and is found to be 42%. AM inhibitor is thus divided between the two separated peaks so that (42-23)/42=45% are bound and 55% are free. Out of the 2.4 mg of AM inhibitor (mol. wt = 7000) taken for this run, 1.1 mg were thus bound to 4.5 mg of trypsin and chymotrypsin (mol. wt = 24,000). This corresponds to a mole to mole binding of AM inhibitor to trypsin and mole to mole binding of AM inhibitor to chymotrypsin. These complexes move with the same s,~ value as the trypsin- or chymotrypsin-AM inhibitor complex.
4. Discussion Anthraniloylation of maleylated BPT inhibitor results in a product which is uniformly labeled on its lysine-15 residue with an anthraniloyl group. The product is heterogeneous as far as maleylation is concerned ; most of it is maleylated on all its primary amino groups only, but some is further maleylated on its hydroxyamino
MODIFIED
INHIBITOR-CHYMOTRYPSlK
COMPLEX
53.5
groups. Maleylated BPT inhibitor on which the lysine-15 group is free has the same inhibitory properties toward trypsin and almost the same toward chymotrypsin as the native inhibitor (Fritz et al., 1969) so that we assume that all the changes introduced in the maleylated inhibitor upon anthraniloylation are due to the anthraniloyl group. The modified inhibitor inhibits both chymotrypsin and trypsin. The inhibition constant for chymotrypsin is measured in a solvent system containing 20% dimethylformamide and 1.6% deoxycholate, and it is therefore impossible to compare the K, Talue for this system with the value in the literature (Fritz et al., 1969) For the maleylated inhibitor, or with the fluorometric interaction constant to which it should be equal, and which is obtained under different conditions. The value for the interaction constant obtained fluorometrically (2 x lo-* M) is similar to the value obtained by Fritz et al. (1969) for the maleylated (Lys-15 free) inhibitor (9x 10e8 M). This might reflect the af6nity of the anthraniloyl group for the tosyl hole w?hich compensates for the disturbed complementarity of the native inhibitor and chymotrypsin. The rise in the inhibition constant for trypsin is enormous. Taking Vincent & Lazdunski’s (1972) recently found value of 6 x 10-l* M for t’he K, of BPT inhibit,or and trypsin, we see that the rise in the K, value upon anthraniloylation is 106-fold. From the fluorescence studies we know that the anthraniloyl group, which is covalent’ly linked to the lysine-15 side-chain, does not interact with trypsin. Thus the big rise in K, must be due to t’he interaction of trypsin wit,h some site other t’han lysina-15. which expresses itself only when lysine-15 is blocked. The conditions of energy transfer are fufilled for the system of tryptophan as a donor and anthraniloyl as an acceptor (Fcrster, 1948 ; Haugland & Stryer, 1967). R,, which is the distance at which the yield of energy t.ransfer is one half, is 20 A for the system tryptophan-anthraniloyl in anthraniloyl chymotrypsin. The fact t’hat energy transfer with a yield of 60% occurs in our system means that the distance between the anthraniloyl group and at least one tryptophan residue is considerably less than R,. If the anthraniloyl group enters the tosyl hole of chymotrypsin upon interaction, a suitable candidate for donor is tryptophan-215 of chymotrypsin, whosr peptide group lines the side of the tosyl hole. Haugland $ Stryer (1967) report t,he same value (65%) for the yield of energy transfer from tryptophan to the anthraniloyl group .in anthraniloyl chymotrypsin. The interaction of AM inhibitor with chymotrypsin is accompanied by an induced asymmetry both in absorption and in emission. Schlessinger & Steinberg (1972) found induced circular dichroism in absorption and induced circular polarization in emission of anthritniloyl chymotrypsin. Both gave negative anisotropy factors, and were of different magnitudes. In the case of the complex AM inhibitor-chymotrypsin, the anisotropy factors are of the same sign and of almost the same magnitude. This would be an indication t,hat bot’h in anthraniloyl chymotrypsin and in our complex the optical activity results from the ont,ranw of the anthraniloyl group into an asymmetric environment. The fact t’hat the maximum of the circular dichroism is shifted towards longer wa,velengths relative to the absorption maximum of anthraniloyl butylamide, led us to think that there is a shift in the absorption spectrum of AM inhibitor upon formation of the complex. Figure 9 shows formation of a shoulder at 325 nm in the absorption spectrum of AM inhibitor upon addition of chymotrypsin. Anthraniloyl butylamide shows a pronounced shift to the red in going from an aqueous solution 3:,
636
Y. ELKANA
(308 nm) to organic solvents (~327 nm) (Elkana, unpublished results), even more pronounced than the shift in absorption maxima found by Haugland & Stryer (1967) for anthranilamide. Such a shift would increase the constant region of the absorption anisotropy factor, ga, and decrease its magnitude, bringing it nearer to ge, the emission anisotropy factor. The anthraniloyl group of AM inhibitor is rigidly held in this asymmetric environment and no change in its position occurs during the lifetime of the excited state, hence the same magnitude for the absorption and emission anisotropy factors. An experiment to prove whether this site is identical with the site into which the anthraniloyl group of anthraniloyl chymotrypsin binds gave no clear answer. No increase in the fluorescence quantum yield of anthraniloyl group was found on addition of anthraniloyl chymotrypsin. Although this is not conclusive evidence, it
i.
L-+-q
300
J
Wavelength
Fra. 9. Absorption spectrum phosphate buffer, pH 7.0.
of AM inhibitor
-C)O
d (nm)
and chymotrypsin,
both at 0.6 x lo- *
M
in 0.1
M-
does not seem likely that two aromatic groups could enter the tosyl hole simultaneously, side by side. The fundamental polarizations of AM inhibitor and of its complex with chymotrypsin are different (Figure 7). The polarization spectra of anthranilamides show two electronic transitions in the long wavelength absorption band (M. Shinitzky, personal communication). Both the shift in absorption and the different p, value of AM inhibitor obtained upon formation of the complex with chymotrypsin might reflect this complexity of the absorption band. At 313 nm we probably excite both electronic transitions to 8 different extent. The ratio of the molecular volumes (7.5) obtained from the polarization measurements is too high. A more plausible value is obtained if we draw the dashed line in Figure 7 by ignoring two experimental points. Such an extrapolation would be compatible with a non-rigidly held transition moment (Wahl & Weber, 1967). For such a case equation (11) should be replaced by the esymptote
where
‘>‘. Pl
PO
The ratio for the volumes thus obtained is 4.35, very close to the ratio of the molecular weights, 31,000 and 7000 for the complex and AM inhibitor, respectively. This treatment implies that the anthraniloyl group has some degree of free rotation about its bond to the free inhibitor, whereas it is rigidly held when it forms the complex with chymotrypsin. Since the difference in the dissociation constant of AM inhibitor with chymotrypsin and trypsin is only fivefold, a triple complex would be detected when equal concentrations of enzymes were taken and if the binding by one of the enzymes did not exclude the binding by the other. It turns out from sedimentation experiments that no such triple complex is formed, although the two separate complexes are formed. AM inhibitor thus has two binding sites, one for chymotrypsin, the anthraniloyl of the lysine-15, and another for trypsin. The two sites are probably close enough t.o each other not to permit the simultaneous formation of a complex. Comparing the inhibition constants of native BPT inhibitor (3 x lo-* M) (Sevilla et al., unpublished results) and the interaction constant of AX inhibitor (2 x lo- 8 M) we must arrive at the conclusion that Iysine-15 is the interaction site of the native inhibitor with chymotrypsin. Otherwise there would have been a site on AM inhibitor, different from lysine-15, which could effectively compete with the Lys-15 site. This competition would have meant that only a fraction of AM inhibitor is responsible for the rise in fluorescence observed in Figure 5. The data in Figure 5, however, can be fitted with curves, calculated according to equation (2), only by taking values [Ilo = 2.35 x 10e7 M and K = 2*5x 10F8 M. The same value of K gives a fair fit. with the emission anisotropy data of Figure 6 when equation (10) is used.
5. Conclusion I am aware of the discrepancy between the results presented above and the inferences from model building of BPT inhibitor-chymotrypsin complex of Blow et al. (1972). It seems to me that the only possible interpretation of the fluorometric result, considering the inhibition of the enzymes by AM inhibitor, is that the anthraniloyl group enters the tosyl hole of chymotrypsin. This may mean that the rest of the AM inhibitor molecule fits the active site of chymotrypsin less tightly than BPT inhibitor. The free energy of interaction of the anthraniloyl group with the tosyl hole might be as high as ~4 kcal mol-l (Inagami, 1964) thus contributing to the total free energy change ~4 kcal mol-l more than the Lys-15 side-chain in BPT inhibitor (Sevilla et al., unpublished data). I would like to acknowledge the assistance of Mr Shaul Yekutiel, Mrs Lydia Schwartz, Mr Peter Yanai, Mrs Josephine Silfen and Mr David Kliger. The measurements of the circular polarization of fluorescence were done by Mr Joseph Schlessinger following very helpful discussions with Professor I. 2. Steinberg and Mr J. Schlessinger.
538
Y.
ELKANA
I am also grateful
for the interest and time spent on the manuscript by Professor J. R. Fresco, Dr H. T. Wright and Dr Meir Shinitzky. I am indebted for constant friendly interest and fruitful discussions to Dr Meir Rigbi in whose laboratory I performed the work. REFERENCES Anderson, G. W., Zimmerman, J. E. & Callahan, F. M. (1964). J. Amer. Chem. Sot. 86, 1839-1842. Azumi, T. & McGlynn, S. P. (1962). J. Chew. Phys. 37, 24132420. Blow, D. M., Wright, C. S., Kukla, D., Ruhlmann, A., Steigemann, W. & Huber, R. (1972). J. Mol. BioZ. 69, 137-144. Butler, P. J. G., Harris, J. I., Hartley, B. S. & Leberman, R. (1969). Biochem. J. 112, 679-689.
Chauvet, J. & Acher, R. (1967). J. Biol. Chem. 242, 4274-4275. Chauvet, J., Nouvel, G. & Acher, R. (1966). Biochim. Biophys. Acta, 115, 130-140. Davis, B. J. (1964). Ann. N.Y. Acad. Sci. 121, 404-427. Forster, T. (1948). Ann. Physik, 2, 55-75. Fritz, H., Schult, H., Meister, R. & Werle, E. (1969). 2. Physiol. Chem. 350, 1531-1540. Gafni, A. & Steinberg, I. Z. (1972). Photochem. Photobiol. 15, 93-96. Green, N. M. & Work, E. (1963). Biochem. J. 54, 257-266. Haas, E., Elkana, Y. & Kulka, R. (1971). Anal. Biochem. 40, 218-226. Habeeb, A. F. S. A. & Atassi, M. Z. (1970). Biochemistry, 9, 4939-4944. Haugland, R. P. & Stryer, L. (1967). In Conformation of Biopolymers (Ramachandran, G. N., ed.), vol. 1, pp. 321-335, Academic Press, London. Hummel, B. C. W. (1959). Can&. J. Biochem. Phyesiol. 37, 1393-1398. Inagami, T. (1964). J. Biol. Chem. 239, 787-791. Jablonski, A. (1960). Bull. Acad. Sci. Ser. Sci. Math. A&. Phya. 8, 259-264. Kraut, H. & Bhargava, N. (1967). 2. Physiol. Chem. 348, 1500-1501. Kress, L. F. & Laskowski, M., Sr (1968). J. BioZ. Chem. 243, 3548-3550. Osterman, L. A. (1971). Anal. B&hem. 43, 254-258. Perrin, F. (1929). Ann. Phys. 12, 169-275. Rigbi, M. (1971). In Proteinase Inhibitors (Fritz, H. & Tschesche, H., eds), pp. 7488, Walter de Gruyter, Berlin. Schlessinger, J. & Steinberg, I. Z. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 769-772. Shaw, E., Mares-Guia, M. & Cohen, W. (1965). Biochemistry, 4, 2219-2224. Sigler, P. B., Blow, D. M., Matthews, B. W. 85 Henderson, R. (1968). -7. Mol. Biol. 35, 143-164.
Steinberg, I. Z. & Gafni, A. (1972). Rev. Sci. In&rum. 43, 409-413. Vincent, J. P. & Lazdunski, M. (1972). Biochemistry, 11, 2967-2977. Wahl, P. & Weber, G. (1967). J. Mol. BioZ. 30, 371-382. Weber, G. (1952). Biochem. J. 51, 145-155. Weber, K. & Osborn, M. (1969). J. BioZ. Chem. 244, 44064412.
(1974) 84, 523-538
Interaction Between the Basic Inhibitor of Bovine Pancreas and Chymotrypsin and Trypsin. Selective Anthraniloylation of the Maleylated Inhibitor YERUDIT
ELKANA
Department of Biological Chemistry The Hebrew University, Jeru,nalem, Irsael (Received 20 September 1973) The interaction between the maleylated basic pancreatic inhibitor, anthraniloylated on its lysine-15 residue, and chymotrypsin is studied by fluorescence intensity, fluorescence polarization, circular dichroism, circular polarization of fluorescence and sedimentation. These measurements show that the interaction takes place through the entrance of the anthraniloyl group into an asymmetric environment in which it is rigidly held. The dissociation constant of the complex is 2.5 x lo-* M. The interaction between the modified inhibitor and trypsin takes place through a site which is not the anthraniloylated lgsine-15 side-chain, yet not far from it.
1. Introduction The interaction between trypsin and the basic pancreatic trypsin inhibitor takes place at the inhibitor residue lysine-15 (Chauvet & Acher, 1967; Kress $ Laskowski, 1968). The site of interaction with chymotrypsin on the intact inhibitor has not been unambiguously proved. Kraut & Bhargava (1967) showed that a complex of trypsin and BPT inhibitort does not inhibit chymotrypsin, whereas the inhibitor itself does, thus suggesting that the site of interaction is either the same as the one for trypsin or is covered by the trypsin area that interacts wit,h the inhibitor. In limited proteolysis experiments, Rigbi (1971) has shown that in the partially reduced inhibitor, lysine-15 and perhaps arginine-39 are loci of attack by chymotrypsin, suggesting that either only the former site or both are the active sites of the native inhibitor toward chymotrypsin. Blow et al. (1972) have shown by model building that the only mode of binding of the inhibitor to chymotrypsin is through the entrance of the side-chain of the inhibitor lysine-15 residue into the specificity pocket of chymotrypsin. In order to get direct proof the role of lysine-15 in the interaction with chymotrypsin, the inhibitor can be labeled at this site with a label which will be sensitive to an interaction with the “tosyl hole” (Sigler et al., 1968) of chymotrypsin. As the inhibitor has no tryptophan residues (Green & Work, 1953), a convenient label is a fluorescent compound capable of accepting energy from the tryptophan residues of chymotrypsin. Haugland & Stryer (1967) labeled chymotrypsin with an anthraniloyl group and found that in the labeled enzyme energy was transferred from the tryptophans to the anthraniloyl group. Although there is no direct Abbreviations used: BPT inhibitor, antbraniloyl (Lys-15 free) maleyleted
basic trypsin BPT inhibitor.
,523
inhibitor
of bovine
pancreas;
AM inhibitor,
524
Y.
ELKANA
evidence that the anthraniloyl is bound to the active site serine-195 of chymotrypsin, it seems likely that the aromatic group is situated in the tosyl hole. Moreover, it was shown by Schlessinger & Steinberg (1972) that bot’h circular dichroism and circular polarization of fluorescence are induced in anthraniloyl-chymotrypsin. In the present papel’ ‘we report studies of the interaction of chymotrypsin and trypsin with the anthraniloyl derivative of maleylated BPT inhibitor. We show that this interaction between the two macromolecules takes place by the entrance of the anthraniloyl group into the tosyl hole of chymotrypsin. Although the modified inhibitor inhibits trypsin, no such interaction between the anthraniloyl group and the active
site of trypsin
can be detected.
2. Materials and Methods N-tosyl-L-phenylalanine chloromethyl-trypsin (lot TRTPCKSIC), a-chymotrypsin (lot CDISLK) and pancreatic trypsin inhibitor compound (lot PICIJB) were purchased from Worthington Bioch&mical Corporation, Freehold, New Jersey. N-tosyl-L-arginine methyl ester was a Miles-Yeda, Rehovot, product. Carbobenzoxy-L-phenylalanine /3-naphthyl ester was prepared by E. Haaa (Haas et aE., 1971). p-Nitrophenyl anthranilate was a gift from A. Carmel (Carmel, unpublished results). Amberlyst A21 was purchased from Serva Feinbiochemica, Heidelberg. N-tosyl-L-lysine chloromethyl ketone-chymotrypsin was prepared according to Shaw et al. (1966). Anthraniloyl-chymotrypsin was prepared according to Haugland & Stryer (1967). The complex of trypsin and BPTinhibitor was purified by passage on Sephadex G75 under the conditions described in the maleylation experiment. Anthranilic acid was recrystallized from water, N-hydroxysuccinimide from ethyl acetate, urea from ethanol and sodium deoxycholate from methanol-ether. Butylamine and dimethylsulfoxide were distilled before use. Maleic anhydride was sublimed. All other mat.erials were of analytical grade. Glass-distilled deionized water was used throughout. Anthranilic acid N-hydroxysucoinimide ester was prepared according to the procedure of Anderson et a.!. (1904). The product was recrystallized 3 times from ethanol. Yield, 86%; m.p., 163’C. Analysis calculated for C11H,004Na: C, 56.41; H, 4.27; N, 11.97. Found: C, 56.03; H, 4.66; N, 12.28. Fluorescence Absorption spectra were taken on a Gary 14 recording speotrophotometer. intensity was measured on an Aminco-Bowman spectrophotofluorometer with J&novia 160 W Xenon or 200 W Xenon-Mercury lamps and an RCAlP28 photomultiplier. Fluorescence polarization measurements were made on the same instrument equipped with Glan prisms supplied by Aminco. Polarization of fluorescence is defined as 2, = (P,, - Pl)/ (E;, + p,), where E;, and FL are the intensities of the light emitted parallel and perpendicular to the direction of polarization of the exciting light. If the polarization is measured in a spectrofluorometer one has to take into account polaxization artifacts introduced by the grating of the emission monochromator. This effect was corrected for according to the method of Azumi & McGlynn (1962) by measuring the grating correction factor, a. Introducing this factor G, p becomes p = (PII - GFJ/(F,, + GPI). F,, and FL were each corrected for the appropriate blank readings. Fluorescence polarization was excited at 313 nm. Temperatures were regulated with a constant temperature accessory. Fluorescence intensity measurements were performed at 25”C, on solutions whose optical density at the wavelength of excitation was below 0.1. Circular di&roism was measured on a Cary 60 spectropolarimeter equipped with a 6002 dichroism aooessory. Measurements of circular polarization of fluorescence were made on an instrument built and described by Steinberg & Gafni (1972). Polyaorylamide gel electrophoreaw were run on a Yeda (Rehovot, Israel) instrument according to Davis (1964) on 11% gels stained with Coomasie Brilliant Blue R (Weber BEOsborn, 1969). Ultrafiltration was carried out with Amicon cells and Di&o membranes. A Beckman model E analytical ultracentrifuge was used for sedimentation studies. Protein hydrolyses were carried out in 6 N-HCl in evacuated sealed tubes for 20 h at
MODIFIED
INHIBITOR-CHYMOTRYPSIN
COMPLEX
525
llO”C, and amino acid analyses were performed on a Beckman amino acid analyzer model 120C. Complex formation between AM inhibitor and chymotrypsin was followed by the increase in fluorescence intensity at 415 run and wss considered complete when the intensity became constant on further addition of chymotrypsin. Peptide mapping of AM inhibitor was carried out on the tryptic hydrolysis of performic acid oxidized AM inhibitor according to Chauvet et al. (1906). The peptides were identified by amino acid analysis. Quantitative compositions could not be obtained owing to the small quantity of peptide in hand and the errors of analysis. The concentration of AM inhibitor and its extinction coefficient in the anthraniloyl absorption band were determined from the amino acid analysis and the absorption spectrum. (a) Anthraniloyl butylamide Butylamine (26 ml, 22 mmol) was added drop-wise to a stirred solution of anthcanilic acid N-hydroxysuccinimide ester (1 g, 4.3 mmol) in ethyl acetate (50 ml). A white precipitate appeared, which was filtered off after stirring the reaction mixture overnight at room temperature. The filtrate wss extracted with a 5% solution of sodium bicarbonate to remove N-hydroxysuccinimide, rinsed 3 times with water and dried over anhydrous sodium sulfate. After evaporation the yield was 0.5 g (60%). The product is amorphous. Analysis calculated for C,,H,,ONz: C, 6875; H, 8.33; N, 1.4.58. Found: C, 68.60; H, 8.20; N, 14.81. (b) MaleyktedfLys-15 free) BPT inhibitor Maleylation of the BPT inhibitor-trypsin complex was carried out according to the procedure of Fritz et aE. ( 1969), with the following modifications : the concentration of the complex was 10 mg/ml ; to a total of 216 mg, 1.5 g of maleic anhydride were added; the final concentration of urea was 6 M and that of ammonium sulfate 25% saturation. The reaction mixture was directly loaded on a Sephadex G75 column (140 cm x 4 cm) and eluted with 0.02 M-ammonium acetate. The yield of maleylated BPT inhibitor was 90%. The calculated degree of maleylation (Butler et al., 1969) was 4.7 -& 0.1 (see Results). (c) Anthranikyl (&/8iTE-15) maleykxted BPT inhibitor (AM inhibitor) 50 mg (210 pmol) of anthranilic acid N-hydroxysuccinimide ester in 5 ml dimethylsulfoxide were added to 40 mg (5-7 ~01) of maleylated (Lys-15 free) BPT inhibitor in 5 ml of 0-l M-triethanolamine buffer, pH 8.0, and the reaction mixture was allowed to stand at room temperature for 48 h. Removal of unreacted and hydrolyzed reagent and solvent wss achieved either by gel filtration through Sephadex G25 or by ultrafiltration through a Diaflo UM2 membrane with 0.02 M-ammonium acetate. In order to separate the desired product from unreacted maleylated BPT inhibitor as well as from traces of BPT inhibitor, Amberlyst A21 (Oeterman, 1971) was used. Preliminary experiments showed that BPT inhibitor was not adsorbed on the resin and that maleylated BPT inhibitor wss eluted at 0.07 N-NaCl and 10 to 15% ethanol. Elution of the desired AM inhibitor from the Amberlyst A21 column (5.5 cm x 1.9 cm) wss carried out by the following steps: loading the desalted reaction mixture and rinsing with 10s3 M-TriseHCl buffer, pH 8.0, containing 0.01 N-NaCl; rinsing with the same buffer containing 1.5 N-NaCl; re-equilibrating with the initial buffer; rinsing with the initial buffer containing 30:/o ethanol. In all these steps elution was continued until no more absorbing materisl could be detected in the eluant. Finally a linear gradient (of a 20-fold vol. excess over the bed resin) of 10V3 M-TriseHCl buffer, pH 8.0, 30% ethanol and 0.01 N to 0.3 N-NaCl was applied. Assay was made by reading the absorbance at 280 nm and at 310 nm snd the fluorescence intensity at 415 nm, excited at 313 run. AM inhibitor was eluted at 0.15 N-Nail, and wss identified by its absorption spectrum (see Results). The fractions containing AM inhibitor were pooled (67% yield), desalted either on Sephadex G25 after reducing the volume by evaporation or, alternatively, by ultrafiltration through a Diaflo UM2 membrane with 0.02 M-ammonium acetate, and lyophilized. The final yield of AM inhibitor wss 50%.
626
Y. ELKANA
(d) Determination of inhibition constant.9 (KI) Inhibition constants (Ki) were determined from Dixon plots, which are obtained by plotting the reciprocal of the initial enzymatic reaction velocity as a function of inhibitor concentration using substrate concentration as a parameter. Reaction mixtures were prepared by incubating the enzyme with the inhibitor at the required concentration for 5 min at 25°C. Reactions were started by adding the substrate to the incubation mixture. Chymotrypsin (final concn 0.1 pg/ml) was assayed with carbobenzoxy-r.-phenylalanine @aphthyl ester as substrate by recording the increase in fluorescence intensity at 420 nm (Haas et al., 1971). The solution contained buffer, 20% dimethylformamide and 1.6% sodium deoxycholate. Trypsin (final concn 0.7 pg/ml) was assayed with tosyl-L-arginine methyl ester by recording the absorbance at 246 nm (Hummel, 1959) on a Gilford model 2000 recording spectrophotometer. As velocities were constant for several minutes, the system was considered to be in a steady state and tho usual inhibition equations are therefore valid. (e) Determination of interaction wnatante (K) Interaction constants between AM inhibitor and chymotrypsin were determined by measuring the increase in the fluorescence of the anthraniloyl moiety of the inhibitor upon addition of chymotrypsin. The measurement was carried out by titrating the inhibitor in the cuvette with increasing amounts of ohymotrypsin. The fluorescence intensity was measured by exciting both at 303 nm and at 313 nm, at a band width of k 5 run, and corrected for readings obtained with the enzyme alone at the same concentrations. The excitation wavelengths were chosen so that at 303 nm tryptophan fluorescence of the enzyme was excited. At 313 nm the only species excited is the anthraniloyl moiety of the inhibitor. The fluorescence intensity (F) is a sum of two contributions, that of the free inhibitor (I) and that of the complexed inhibitor (EI) :
F = 411 + B[EIl, where CLand /3 are constants independent inn the relations
of the concentrations
[I] = [I],, - [EI] ([El is the concentration F = a[Ik, + -$[Elo ‘-” The subscript Differentiating
(1) of the components.
Insert-
[II [El
and K = [EIl
of free enzyme) into equation
+ mo + K-d/(([Elo
(1) we obtain:
+ VI0 + w
- 4~w0m0~1.
0 denotes total concentrations. and taking the limit at [El, -+ 0 we get
The quantity (/I- a) [Ilo can be obtained from the graph of F vemua [El, by subtracting the initial value from the final value at the plateau. The interaction constant K can thus be obtained directly from the graph. (f) Determination of the e$?&ncy of energy transfer Let us designate the light intensity at the wavelength r\ as L(h), the fluorescence quantum yield as Qr, the yield of energy transfer as & and the extinction coefficients of AM inhibitor and chymotrypsin as eAMi(A) and C&A), respectively. The two constants aandjlwillbegivenby: a = L(~Mu&) (4) and
B = uwPxlIwdI(~) Obvhdy
at h = 313 nm, scT = 0.
+ ~dv~tl.
(5)
MODIFIED In reality
INHIBITOR-CHYMOTRYPSIN
the exciting
light
is not monochromatic,
COMPLEX and equations
(4) and (5) change into (4’)
a = #:sQ A)u&)d~
and
B = 4iVU~)~mhWX
527
+ 4,SWV4Nd~l.
The value j3ia is taken from the AM inhibitor titration curve by chymotrypsin given by the ratio of the fluorescence values at the plateau to the initial value. equations (4) and (5) we get for A = 313 run,
(5’) and is From
_s c,"' $'f, 0 =-=
(6)
(3,,,= d’r(l+ g A)= (gJ1 + g A).
(7)
a
313
4:
and for 303 urn
For non-monochromatic
light,
equations
3.
(4’) and (5’) have
to be used.
Results
(a) Ckmacterization of anthraniloyl
(Lys-15) mdeylated inhibitor
AM inhibitor was eluted as the only peak on rechromatography on Amberlyst A21 with a salt gradient in 30% ethanol containing lop3 M-Tris+HCl buffer, pH 8.0, at 0.15 N-NC&~ (Fig. 1). The rechromatographed AM inhibitor, which was desalted and lyophilized, gave one main fluorescent band, one weak band, and several very weak bands on polyacrylamide gel electrophoresis (Plate I). The weak bands can be attributed to inhibitor species that were maleylated to a different extent on their hydroxyamino acid residues (Habeeb & Atassi, 1970), but were too weak to be visible as fluorescent bands.
Fio. 1. Rechromatography of AM inhibitor on Amberlyst AZ1 in 10e3 M-Triu.HCl [N&l]; --o---O-, pH 8.0, 30% ethanol and a salt gradient as indicated. -O--e--, F;;;. --C---o--, A,,,; --a-A----,
buffer, Azso;
528
Y.
ELKANA
The absorption spectrum of (0.75-&0.05) x lob4 M-m inhibitor is given in Figure 2. It shows absorption typical of the anthraniloyl moiety in the wavelength region above 300 nm and typical of the maleylated inhibitor in the region below 300 nm. The extinction coefficient of AM inhibitor at 305 nm is 2950&200 cm2 mmol-I, which is equal to that of anthraniloyl butylamide. For comparison, the spectrum of equimolar concentrations of maleylated BPT inhibitor and anthraniloyl butylamide, at (OG35f0.15) x 10M4 M, is given in the same Figure. The spectra are identical. The fluorescence spectrum of AM inhibitor is given in Figure 8 and is identical to that of anthraniloyl butylamide. From the peptide map of AM inhibitor (Fig. 3) and the list of the expected peptides (Table l), we see that trypsin cleaved only after the endo arginine residues. The only fluorescent spot, that of peptide (1+2), gave no ninhydrin colour, in accordance with a blocked N-terminal and a fluorescent group on lysine-15. Peptide (7+8), which contains the only serine residue of AM inhibitor, appeared twice, probably owing to partial maleylation of the serine residue (Habeeb BEAtassi, 1970), and the resulting greater mobility in a positive direction. Two very weak ninhydrin positive spots could be detected, but did not reveal any peptides by amino acid analysis. These results are consistent with the multiple bands appearing in gel
Wavelength (nml
FIG. 2. Absorption spectra of 0.76 x IO- 4 M-AM inhibitor in 0.1 ~-ammonium acetate (----L of 0.86 x lo- * m-anthraniloyl butylamide (* . * . * . * . e), and of 0,86x lo- 4 x-anthreniloyl butylemide + 0.85 x lo-’ M-meleylated BPT inhibitor (- . - . ) in 0.1 iw-phosphate buffer, pH 7.5.
sr T c
MODIFIED
INHIBITOR-CHYMOTRYPSIN
529
COMPLEX
TABLE 1 Expected tryptic peptides of oxidized AM inhibitor Sequence
Peptides?
1 A g-Pro-Asp-Phe-CysO,H-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro17 16 CysOsH-Lys-Ala-Arg 18 20 Ile-Ile-Arg 21 Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-CysO,H-Glu-Thr39 Phe-Val-Tyr-Gly-Gly-CysOsH-Arg 40 42 Ala-Lys-Arg 43 Asn-Am-Phe-Lys-Ser-Ala-Glu-Asp-CysOsH-MetOssArg 54 68 Thr-CysOsH-Gly-Gly-Ale
I+2
3 4+5
6 7+8 9
t Numbered
according
to Chsuvet
53
et al. (1960).
electrophoresis, and the somewhat high calculated degree of maleylation, 4.7, for one N-terminal and three c-amino groups. The circular dichroism spectra of BPT and AM inhibitors are identical. ;lu’o gross conformational change has taken place in the inhibitor upon modification. AM inhibitor ,+hows no optical activity in the anthraniloyl absorption band.
7.
I-
i
7t0 ,_----> ‘.___A
,/---> ‘./’
1
4t5 =E
,_----. : *-_-_s,
23
6
Fm. 3. Peptide mapping of the tryptic digest of performic acid oxidized AM inhibitor. Peptides are numbered according to Chauvet et al. (1906). Solid outline, ninhydrin positive; dashed outline, weak ninhydrin positive ; hatched area, ninhytlrin negative, fluorescent.
Y.
530
(b) Interaction
of AM
ELKANA
inhibitor
with chynwtrypsin
and trypsin
Figure 4 shows Dixon plots of the inhibition of u-chymotrypsin and trypsin. The K, values of the competitive inhibitions are (3fl) x 10d7 M and (l-3&0.2) x 10V7 M, respectively, for chymotrypsin and trypsin. (i) Fluorivnetric
study of the interaction
between chymotrypsin.
and AM
inhibitor
Typical titration curves of AM inhibitor with chymotrypsin are shown in Figure 5. Both were measured at identical concentrations of AM inhibitor but excited at different wavelengths. We see that there is a rise in fluorescence intensity of the anthraniloyl moiety of AM inhibitor when excited both at its absorption band and at the aromatic absorpt’ion band of chymotrypsin. This rise eventually reaches a plateau, presumably when all the AM inhibitor is bound to the chymotrypsin. The rise in intensity is almost sevenfold when t,he fluorescence is excited in the chymotrypsin tryptophan band and half this value when excited in the anthraniloyl absorption band. There is no shift in wavelength of maximum intensity. From t,he titration curves of the fluorescence intensity as a function of enzyme concentration and by using equation (3), we get K = (2&l) x 1O-8 M. By using equations (6) and (7) and the data of Figure 5 we obtain an energy transfer efficiency of (60-&5)% from the tryptophans of chymotrypsin to t’he ant,hraniloyl group of AM inhibitor upon formation of the complex. No change in anthraniloyl fluorescence intensity is observed when trypsin is added to AM inhibitor (Fig. 5). (ii) Polarization
studies on the formation
of the AM
inhibitor-chymotrypsin
Figure 6 shows the rise in emission anisotropy of AM inhibitor chymotrypsin concentration. Emission anisotropy, r, is defined
complex a function of as (Pii - F,)/
as
T-
-3
-2
-I
0
I
2
3
. -3
-2
-I
0
I
2
3
[AM inhibitor1 (M x 107) (a)
(b)
4. Dixon plots for the inhibition Substrate concentrations were : Fro.
-o-o---, --A--A-, -v--v-,
&ails of assay we in Met&& d in fluorescence arbitrary
by AM inhibitor
of trypsin
(a) and of chymotrypsin
1.1 x 10-4 M; -*--a--, 0.3 x 10-d M; 2-7x lo-* M; --A-A--, 0.8x 10-4 rd; 6.7 x lo- 4 ?a; ---‘I-v---, 1 x10-4 M. and Methods. v is expressed in units of dRacB nm min-1 units min-’ for chymotrypsin.
(b).
for trypsin
MODIFIED
0 Li 0
INHIBITOR-CHYMOTRYPSIN
A-.. LI_II_LuI.-.L I
2
3
4
COMPLEX
531
I I I I ! 1..LLLLL
5
6
[El,
7
8
9
IO
II
(M x10-‘)
Fra. 6. Fluorescence intensity of 2.35 x 10e7 M-Ah% inhibitor as a function of chymotrypsin A) and trypsin (0, n ) concentrations, in 0.04 M-Tris.HCl buffer, pH 8.0. ( l , (1) Excited at 303 nm; (A, A) excited at 313 nm, and normalized at [El, = 0. The curves are calculated according to equation (2) with K = 2.5 x 10e8 M. (0,
(F, + 2F,) (Jablonski, 1960). We see that the emission anisotropy rises as a function of [El,, event)ually reaching a constant level. The functional dependence of T on [El,, is different from that of P on [El,. The anisotropy of a system that is composed of several components is an additive function of the individual anisotropies, ri (Jablonski, 1960) : i
where fi is the fraction of the fluorescence intensity ponent. Equation (8) will take the form :
for the system AM inhibitor-chymotrypsin.
contributed
by the ith com-
The superscripts I and EI denote free
FIG. 8. Emission anisotropy of 2.36 x lo-’ M-AM inhibitor a~ a function of chymotrypsin concentration in 0.04 iv-Tris *HCl buffer, pH 8.0, at 16°C. The curve is calculated a.ocording to equation (X0) with K = 2.6 x 10e8 M.
532
P. ELKANA
and bound inhibitor, respectively. and R, equation (9) becomes
By expressing [I 1 and [El] in terms of [l IS. i E j.
TE’$‘f fvlo
+
?-=
[I]
2
0+
24’f -
?-I
Wlo
1 Plo
+ [Ilo -t K - 2/Wlo + [Ilo + K - &~:I,
f- lllo +- W + PI, + W
- 4l3l,[ll,)l - 4CEloUlo)J (10)
Figure 7 shows the dependence of the degree of polarization of AM inhibitor and of AM inhibitor-chymotrypsin complex on T/T (T, the absolute temperature and 7, the viscosity of the solution). The measurements were carried out isot,hermally at, 20°C on solutions of varying sucrose concentrations. The complex gives a straight line with an intercept at l/p = 3.6 and a slope of 1.3 x 10m4. The polarization values of AM inhibitor are very low, and the experimental error in their measurement is large. A. least squares calculation gives an intercept of 8.2 and a slope of 6*9 x 10m4 for the AM inhibitor polarization. The Perrin-Weber formula for the degree of polarization in the case of a transition moment rigidly held to the molerule is
(11) (Perrin, 1929; Weber, 1952). p, is the fundamental polarization, 7 the decay time of the fluorescence, k the Boltzmann constant and v the molecular volume. If we use this relation, the measured intercepts and slopes of Figure 7 and the relation
we get @I -UI = 7-5.
FIG. 7. Perrin plot of AM inhibitor (0) and of the complex AM inhibitor-chymotrypsin (A) (both at 2.35 IO-’ rd) in aqueous solutions rtt 20°C. Viscosities were taken from the Handbook of Chemistry and Physics, 44th edn.
x
sucr088
MODIFIED
INHlBITOR-CHYMOTRYPSIN
COMPLEX
5 3 :I
(iii) Circular dichroism and circular polarization of jluorescence of the AM inhibitoru-ch ymot ypsin complex The complex between AM inhibit,or and cc-chymotrypsin exhibits induced asymmetry both in absorption and in emission. A positive band appears in the circular dichroism spectrum of the complex in the region of the anthraniloyl absorption. There is also a circularly polarized component in the emission band of the complex. These effect,s are expressed as anisotropy fact’ors (not to he confused with Jablonski’s emission. anisotropy) : for absorption eL-.sR g, = -=E
de F
and for emission gl?=
FL--FP, F/2
AF = F/2’
The subscripts L and R denote left and right-hand circularly polarized components, respectively (Gafni & Steinberg, 1972; Schlessinger & Steinberg, 1972). In Figure 8 1)oth the absorption and the emission anisotropy factors are shown with the absorpt,ion spectrum of anthraniloyl butylamide and the corrected fluorescence and circular dichroism spectra of the complex. We see that the circular dichroism band has a maximum of 325 nm and is shifted by 20 nm to the red relative to the absorption spectrum of anthraniloyl butylamide. The absorption anisotropy band is constant over most of the emission band. Both factors are positive and of similar magnitudes. The complex of AM inhibitor and hrypsin shows no circular dichroism in the ant,hraniloyl absorption band.
FIG. 8. Spectroscopic data for AM inhibitor-cc-chymotrypsin complex. ), Anthreniloyl butylamide absorption spectrum in 0.1 M-phosphate buffer, pH 7.6; AM inhibitor and AM inhibitor-chymotrypsin complex fluoresoence spectra in 0.04 buffer, pH 8.0; -*-.-, circular dichroism of complex expressed 88 de = eL - cR AE in 0.04 :M-Tris.HCl buffer, pH 8.0; -m-e--, absorption anistropy factor, ga = -, of aomplex ; E AF .-, ‘\,--.. :‘, --, emission anisotropy factor, qe - -, of complex. F/2
( --___ (--------), Ma-Tris*HCl
Y.
534
ELKANA
(iv) Interaction of AM inhibitor with anthraniloyl chymotrypsin When AM inhibitor is titrated with anthraniloyl chymotrypsin in the same manner as wit(h chymotrypsin, the rise in fluorescence is proportional to the concentration of anthraniloyl chymotrypsin. From this type of experiment it seems thab no inberaction takes place between the modified inhibitor and the modified enzyme. (v) Sedimentation experiments Table 2 gives the sZo values of AM inhibitor, of a mixture of trypsin and chymotrypsin and of t,he ternary mixture of AM inhibit,or, chymotrypsin and trypsin. In the last experiment (shown in Plat’e II) t,he amount of AM inhibitor exceeded the maximum amount which could have bound to bot’h trypsin and chymotrypsin. In that run, the peak separated into t,wo, one sedimenting with (3.0&0*1) S and the other with 1.4 S. From the ratio between t,he areas of the separated peaks we calculate that the amount of free AM inhibitor is 237” of the total amount of the material in this run. The fraction of total AM inhibitor, bound and free, in this mixture is TABLE 2 Sedimentation
coeficients of AM khibitor, and of their ternary
Run
oj’ trypsin
Composition (in 1 ml) AM inhibitor 2.6 mg trypsin 2 mg chymotrypsin 2.4 mg AM inhibitor 2.6 mg trypsin 2 mg chymotrypsin
Experiments were run in 0.04 BI Tris.HCl buffer, in 0.02 rd.ammonium aoetete, at 59,780 revs/min.
and chyrrwt,:ypsitL,
mixture G20(IkO.1) (S) 1.6 2.9
1.4 and
3.9
pH 8.0, except for AM inhibitor
whioh was
calculated from comparison of the Schlieren patterns at the beginnings of run numbers 2 and 3, where equal amounts of chymotrypsin and trypsin are taken, and is found to be 42%. AM inhibitor is thus divided between the two separated peaks so that (42-23)/42=45% are bound and 55% are free. Out of the 2.4 mg of AM inhibitor (mol. wt = 7000) taken for this run, 1.1 mg were thus bound to 4.5 mg of trypsin and chymotrypsin (mol. wt = 24,000). This corresponds to a mole to mole binding of AM inhibitor to trypsin and mole to mole binding of AM inhibitor to chymotrypsin. These complexes move with the same s,~ value as the trypsin- or chymotrypsin-AM inhibitor complex.
4. Discussion Anthraniloylation of maleylated BPT inhibitor results in a product which is uniformly labeled on its lysine-15 residue with an anthraniloyl group. The product is heterogeneous as far as maleylation is concerned ; most of it is maleylated on all its primary amino groups only, but some is further maleylated on its hydroxyamino
MODIFIED
INHIBITOR-CHYMOTRYPSlK
COMPLEX
53.5
groups. Maleylated BPT inhibitor on which the lysine-15 group is free has the same inhibitory properties toward trypsin and almost the same toward chymotrypsin as the native inhibitor (Fritz et al., 1969) so that we assume that all the changes introduced in the maleylated inhibitor upon anthraniloylation are due to the anthraniloyl group. The modified inhibitor inhibits both chymotrypsin and trypsin. The inhibition constant for chymotrypsin is measured in a solvent system containing 20% dimethylformamide and 1.6% deoxycholate, and it is therefore impossible to compare the K, Talue for this system with the value in the literature (Fritz et al., 1969) For the maleylated inhibitor, or with the fluorometric interaction constant to which it should be equal, and which is obtained under different conditions. The value for the interaction constant obtained fluorometrically (2 x lo-* M) is similar to the value obtained by Fritz et al. (1969) for the maleylated (Lys-15 free) inhibitor (9x 10e8 M). This might reflect the af6nity of the anthraniloyl group for the tosyl hole w?hich compensates for the disturbed complementarity of the native inhibitor and chymotrypsin. The rise in the inhibition constant for trypsin is enormous. Taking Vincent & Lazdunski’s (1972) recently found value of 6 x 10-l* M for t’he K, of BPT inhibit,or and trypsin, we see that the rise in the K, value upon anthraniloylation is 106-fold. From the fluorescence studies we know that the anthraniloyl group, which is covalent’ly linked to the lysine-15 side-chain, does not interact with trypsin. Thus the big rise in K, must be due to t’he interaction of trypsin wit,h some site other t’han lysina-15. which expresses itself only when lysine-15 is blocked. The conditions of energy transfer are fufilled for the system of tryptophan as a donor and anthraniloyl as an acceptor (Fcrster, 1948 ; Haugland & Stryer, 1967). R,, which is the distance at which the yield of energy t.ransfer is one half, is 20 A for the system tryptophan-anthraniloyl in anthraniloyl chymotrypsin. The fact t’hat energy transfer with a yield of 60% occurs in our system means that the distance between the anthraniloyl group and at least one tryptophan residue is considerably less than R,. If the anthraniloyl group enters the tosyl hole of chymotrypsin upon interaction, a suitable candidate for donor is tryptophan-215 of chymotrypsin, whosr peptide group lines the side of the tosyl hole. Haugland $ Stryer (1967) report t,he same value (65%) for the yield of energy transfer from tryptophan to the anthraniloyl group .in anthraniloyl chymotrypsin. The interaction of AM inhibitor with chymotrypsin is accompanied by an induced asymmetry both in absorption and in emission. Schlessinger & Steinberg (1972) found induced circular dichroism in absorption and induced circular polarization in emission of anthritniloyl chymotrypsin. Both gave negative anisotropy factors, and were of different magnitudes. In the case of the complex AM inhibitor-chymotrypsin, the anisotropy factors are of the same sign and of almost the same magnitude. This would be an indication t,hat bot’h in anthraniloyl chymotrypsin and in our complex the optical activity results from the ont,ranw of the anthraniloyl group into an asymmetric environment. The fact t’hat the maximum of the circular dichroism is shifted towards longer wa,velengths relative to the absorption maximum of anthraniloyl butylamide, led us to think that there is a shift in the absorption spectrum of AM inhibitor upon formation of the complex. Figure 9 shows formation of a shoulder at 325 nm in the absorption spectrum of AM inhibitor upon addition of chymotrypsin. Anthraniloyl butylamide shows a pronounced shift to the red in going from an aqueous solution 3:,
636
Y. ELKANA
(308 nm) to organic solvents (~327 nm) (Elkana, unpublished results), even more pronounced than the shift in absorption maxima found by Haugland & Stryer (1967) for anthranilamide. Such a shift would increase the constant region of the absorption anisotropy factor, ga, and decrease its magnitude, bringing it nearer to ge, the emission anisotropy factor. The anthraniloyl group of AM inhibitor is rigidly held in this asymmetric environment and no change in its position occurs during the lifetime of the excited state, hence the same magnitude for the absorption and emission anisotropy factors. An experiment to prove whether this site is identical with the site into which the anthraniloyl group of anthraniloyl chymotrypsin binds gave no clear answer. No increase in the fluorescence quantum yield of anthraniloyl group was found on addition of anthraniloyl chymotrypsin. Although this is not conclusive evidence, it
i.
L-+-q
300
J
Wavelength
Fra. 9. Absorption spectrum phosphate buffer, pH 7.0.
of AM inhibitor
-C)O
d (nm)
and chymotrypsin,
both at 0.6 x lo- *
M
in 0.1
M-
does not seem likely that two aromatic groups could enter the tosyl hole simultaneously, side by side. The fundamental polarizations of AM inhibitor and of its complex with chymotrypsin are different (Figure 7). The polarization spectra of anthranilamides show two electronic transitions in the long wavelength absorption band (M. Shinitzky, personal communication). Both the shift in absorption and the different p, value of AM inhibitor obtained upon formation of the complex with chymotrypsin might reflect this complexity of the absorption band. At 313 nm we probably excite both electronic transitions to 8 different extent. The ratio of the molecular volumes (7.5) obtained from the polarization measurements is too high. A more plausible value is obtained if we draw the dashed line in Figure 7 by ignoring two experimental points. Such an extrapolation would be compatible with a non-rigidly held transition moment (Wahl & Weber, 1967). For such a case equation (11) should be replaced by the esymptote
where
‘>‘. Pl
PO
The ratio for the volumes thus obtained is 4.35, very close to the ratio of the molecular weights, 31,000 and 7000 for the complex and AM inhibitor, respectively. This treatment implies that the anthraniloyl group has some degree of free rotation about its bond to the free inhibitor, whereas it is rigidly held when it forms the complex with chymotrypsin. Since the difference in the dissociation constant of AM inhibitor with chymotrypsin and trypsin is only fivefold, a triple complex would be detected when equal concentrations of enzymes were taken and if the binding by one of the enzymes did not exclude the binding by the other. It turns out from sedimentation experiments that no such triple complex is formed, although the two separate complexes are formed. AM inhibitor thus has two binding sites, one for chymotrypsin, the anthraniloyl of the lysine-15, and another for trypsin. The two sites are probably close enough t.o each other not to permit the simultaneous formation of a complex. Comparing the inhibition constants of native BPT inhibitor (3 x lo-* M) (Sevilla et al., unpublished results) and the interaction constant of AX inhibitor (2 x lo- 8 M) we must arrive at the conclusion that Iysine-15 is the interaction site of the native inhibitor with chymotrypsin. Otherwise there would have been a site on AM inhibitor, different from lysine-15, which could effectively compete with the Lys-15 site. This competition would have meant that only a fraction of AM inhibitor is responsible for the rise in fluorescence observed in Figure 5. The data in Figure 5, however, can be fitted with curves, calculated according to equation (2), only by taking values [Ilo = 2.35 x 10e7 M and K = 2*5x 10F8 M. The same value of K gives a fair fit. with the emission anisotropy data of Figure 6 when equation (10) is used.
5. Conclusion I am aware of the discrepancy between the results presented above and the inferences from model building of BPT inhibitor-chymotrypsin complex of Blow et al. (1972). It seems to me that the only possible interpretation of the fluorometric result, considering the inhibition of the enzymes by AM inhibitor, is that the anthraniloyl group enters the tosyl hole of chymotrypsin. This may mean that the rest of the AM inhibitor molecule fits the active site of chymotrypsin less tightly than BPT inhibitor. The free energy of interaction of the anthraniloyl group with the tosyl hole might be as high as ~4 kcal mol-l (Inagami, 1964) thus contributing to the total free energy change ~4 kcal mol-l more than the Lys-15 side-chain in BPT inhibitor (Sevilla et al., unpublished data). I would like to acknowledge the assistance of Mr Shaul Yekutiel, Mrs Lydia Schwartz, Mr Peter Yanai, Mrs Josephine Silfen and Mr David Kliger. The measurements of the circular polarization of fluorescence were done by Mr Joseph Schlessinger following very helpful discussions with Professor I. 2. Steinberg and Mr J. Schlessinger.
538
Y.
ELKANA
I am also grateful
for the interest and time spent on the manuscript by Professor J. R. Fresco, Dr H. T. Wright and Dr Meir Shinitzky. I am indebted for constant friendly interest and fruitful discussions to Dr Meir Rigbi in whose laboratory I performed the work. REFERENCES Anderson, G. W., Zimmerman, J. E. & Callahan, F. M. (1964). J. Amer. Chem. Sot. 86, 1839-1842. Azumi, T. & McGlynn, S. P. (1962). J. Chew. Phys. 37, 24132420. Blow, D. M., Wright, C. S., Kukla, D., Ruhlmann, A., Steigemann, W. & Huber, R. (1972). J. Mol. BioZ. 69, 137-144. Butler, P. J. G., Harris, J. I., Hartley, B. S. & Leberman, R. (1969). Biochem. J. 112, 679-689.
Chauvet, J. & Acher, R. (1967). J. Biol. Chem. 242, 4274-4275. Chauvet, J., Nouvel, G. & Acher, R. (1966). Biochim. Biophys. Acta, 115, 130-140. Davis, B. J. (1964). Ann. N.Y. Acad. Sci. 121, 404-427. Forster, T. (1948). Ann. Physik, 2, 55-75. Fritz, H., Schult, H., Meister, R. & Werle, E. (1969). 2. Physiol. Chem. 350, 1531-1540. Gafni, A. & Steinberg, I. Z. (1972). Photochem. Photobiol. 15, 93-96. Green, N. M. & Work, E. (1963). Biochem. J. 54, 257-266. Haas, E., Elkana, Y. & Kulka, R. (1971). Anal. Biochem. 40, 218-226. Habeeb, A. F. S. A. & Atassi, M. Z. (1970). Biochemistry, 9, 4939-4944. Haugland, R. P. & Stryer, L. (1967). In Conformation of Biopolymers (Ramachandran, G. N., ed.), vol. 1, pp. 321-335, Academic Press, London. Hummel, B. C. W. (1959). Can&. J. Biochem. Phyesiol. 37, 1393-1398. Inagami, T. (1964). J. Biol. Chem. 239, 787-791. Jablonski, A. (1960). Bull. Acad. Sci. Ser. Sci. Math. A&. Phya. 8, 259-264. Kraut, H. & Bhargava, N. (1967). 2. Physiol. Chem. 348, 1500-1501. Kress, L. F. & Laskowski, M., Sr (1968). J. BioZ. Chem. 243, 3548-3550. Osterman, L. A. (1971). Anal. B&hem. 43, 254-258. Perrin, F. (1929). Ann. Phys. 12, 169-275. Rigbi, M. (1971). In Proteinase Inhibitors (Fritz, H. & Tschesche, H., eds), pp. 7488, Walter de Gruyter, Berlin. Schlessinger, J. & Steinberg, I. Z. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 769-772. Shaw, E., Mares-Guia, M. & Cohen, W. (1965). Biochemistry, 4, 2219-2224. Sigler, P. B., Blow, D. M., Matthews, B. W. 85 Henderson, R. (1968). -7. Mol. Biol. 35, 143-164.
Steinberg, I. Z. & Gafni, A. (1972). Rev. Sci. In&rum. 43, 409-413. Vincent, J. P. & Lazdunski, M. (1972). Biochemistry, 11, 2967-2977. Wahl, P. & Weber, G. (1967). J. Mol. BioZ. 30, 371-382. Weber, G. (1952). Biochem. J. 51, 145-155. Weber, K. & Osborn, M. (1969). J. BioZ. Chem. 244, 44064412.