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Pepsin inhibition by a high specific activity radioiodinated derivative of pepstatin
ARCHIVES OFBIOCHEMISTRYANDBIOPHYSICS Vol. 194, No. 1, April 15, pp. 157-164, 1979
Pepsin inhibition
by a High Specific Activity Derivative of Pepstatinl
ROBERT JAY WORKMAN’ Veteraw
Administration Received
Hospital, September
AND
Radioiodinated
DONNA W. BURKITT
1310 24th Avenue South, Nashville,
Tennessee SROd
18, 1978; revised October 11. 1978
High specific activity radioiodinated derivatives of pepstatin which retain the inhibitory properties of the parent compound toward pepsin have been prepared. Pepstatin was first coupled to L-tyrosine methyl ester or L-3-iodotyrosine methyl ester. Pepstatin-Ltyrosine methyl ester was iodinated with lz51 and chloramine-T and purified to homogeneity on LH20 columns. Nonradioactive pepstatin-L-iodotyrosine methyl ester and pepstatinL-[1251]monoiodotyrosine methyl ester coeluted in gel filtration column chromatography. Using thin layer chromatography and radioautography a mixture of both these compounds also migrated as a single spot. On the basis of these criteria the radioiodinated pepstatin was judged to be virtually pure and have a specific activity of 2.16 Ci/pmol. Using the digestion of hemoglobin to assay peptic activity at pH 2.0, both pepstatin and pepstatinL-iodotyrosine methyl ester demonstrated similar inhibition of pepsin, indicating that the addition of the iodotyrosine methyl ester group to the carboxyl terminus of pepstatin did not alter the inhibitory properties of the parent compound. By means of equilibrium dialysis at pH 5.0 in which pepstatin-L-[‘2jI]monoiodotyrosine methyl ester was used in trace quantities, the dissociation constants K, of pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester were determined and found to be 4.57 x 10-l’ _f 1.42 M and 1.31 x 10-l’ ? 0.63 M respectively. The closeness of these values to each other provides additional quantitative evidence that pepstatin-L-iodotyrosine methyl ester reacts with pepsin in a manner similar to that observed with native, unmodified pepstatin and suggests that pepstatin-L-[““Ilmonoiodotyrosine methyl ester may be used as an affinity label of acid proteases under the same circumstances in which native pepstatin is known to react with this class of enzymes.
The most potent known inhibitor of the so-called acid protease class of enzymes is the bacterial fermentation product pepstatin (isovaleryl-L-valyl-L-valyl-4-amino3-hydroxy-6-methyl heptanoyl-L-alanyl-4amino-3-hydroxyl-6-methyl heptanoic acid). Due to its potency this substance has found numerous applications in studying the biochemistry of the enzymes which it inhibits, most notably pepsin (1, 9-11, 20, 21), cathepsin D (1, 2, 3, 8, 22), renin (1, 4, 5, 11, 13, 17), and fungal acid proteases (20, 21). Several of these studies have been facilitated by utilizing radioactive derivatives of pepstatin labeled with “C or 3H. Kunimoto ’ This investigation was supported by the Medical Research Service of the Veterans Administration, * To whom correspondence should be addressed.
(9, 10) studied the binding of pepstatin to pepsin using tritiated pepstatin of specific activity 68 ~Cil~mol prepared by exposing pepstatin to tritium gas. 14C-Labeled pepstatins with a maximum specific activity of 164 &X/mmol were obtained by Morishima et al. (14) by incubating cultures of Streptomyces testaceus with 14C-labeled amino acids and isolating the pepstatins released into the culture broth. Knight and Barrett (8) coupled pepstatin to [3H]glycine to obtain a product with a specific activity of 31 &i/pmol which was at least 50% impure since it contained both uncoupled pepstatin and some other unidentified contaminant which did not bind to cathepsin D. These radiolabeled pepstatins possessed low specific activity and in the last report the product was impure. The present study describes et al.
157
0003.9861/79/050157-08$02.00/0 Copyright G 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
158
WORKMAN AND BURKITT
the preparation of a radioiodinated pepstatin derivative of very high specific activity and purity which retains the inhibitory properties of the parent compound. The applicability of such a compound is illustrated by using the radioiodinated pepstatin in trace quantities at an accurately known specific activity to determine the dissociation constants of pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester. EXPERIMENTAL
PROCEDURE
Materials Pepstatin A was a gift from H. Umezawa (Tokyo, Japan). Porcine pepsin, twice crystallized (specific activity 4500 U/mg of protein; lot SSC-SOSO), L-tyrosine methyl ester, and L-3-iodotyrosine were from Sigma Chemical Company, St. Louis, Missouri. Dicyclohexylcarbodiimide, chloramine-T, cellulose tic sheets with fluorescent indicator and no-screen medical X-ray film NSZT were from the Eastman Kodak Company, Rochester, New York. Bovine hemoglobin, twice crystallized, was obtained from Miles Laboratories, Inc., Kankakee, Illinois. Spectral grade solvents were from J. T. Baker, Phillipsburg, New Jersey. Sulfuric acid-dichromate spray and 3 N methanol&HCl were from Supelco, Inc., Bellefonte, Pennsylvania. LH20 was obtained from Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey. Silicar CC-4 was from Mallinckrodt, Inc., St. Louis, Missouri, Carrier-free high concentration (17 Ciimg) sodium [1251]iodidewas obtained from New England Nuclear, Boston, Massachusetts. Merck precoated tic silica gel 60 F254 sheets of 0.24 mm-thickness with fluorescent indicator on aluminum backing were obtained through Brinkmann Instruments, Inc., Des Plaines, 111. Other reagent grade chemicals were obtained from various commercial sources.
Methods Thin layer chromatography (tic). The following tic solvent systems3 were used: (A) chloroform:methanol:acetic acid, 86:9:5; (B) chloroform:methanol: ammonia, 75:24:1; (C) ethyl acetate:pyridine:acetic acid:water, 35:35:7:14; (D) isopropanol:ethyl acetate: 6 N ammonia:acetone, 35:30:25:20. Solvents A and B were used with silica gel tic plates on aluminum 3 Abbreviations used: Solvent A, chloroform:methanobacetic acid, 86:9:5; solvent B, chloroform:methanol: ammonia, 75:24:1; solvent C, ethyl acet.ate:pyridine: acetic acid:water, 35:35:7:14; solvent D, isopropanol: ethyl acetate:6 N ammonia:acetone, 35:30:25:20; E, total counts; e, enzyme concentration.
backing. Solvents C and D were used with cellulose tic plates on plastic. After performing tic of radioiodinated compounds, the tic plate was pressed against Kodak no-screen X-ray film to produce a radioautograph. The coating of appropriate tic plates was scraped off and counted in a well scintillation counter (Searle Model 1197, efficiency 78%) to quantitate the actual radioactivity in each spot seen on radioautography. Coupling of’ pepstatin to tyrosine esters. L-3-Iodotyrosine was esterified with 3 N methanolic-HCl and purified on LH20 (1 x 22 cm) in 50% methanolwater. Purity of the product was confirmed by tic in solvent system B. L-tyrosine methyl ester (known) and L-iodotyrosine methyl ester had an R, of 0.989, whereas the free acid had an R, of 0.473. Pepstatin, tyrosine methyl ester, and dicyclohexylcarbodiimide in molar proportions of approximately 1:4:4 were suspended in 8-10 ml of dichloromethane and stirred at room temperature for 96 h. The solvent was removed by evaporation, and the residue was redissolved in methanol and eluted from an LH20 column (1 x 110 cm) in methanol by gravity flow through a uv flow cell (ISCO Model UA5 with Type 6 optical unit) at 280 nm. Products were identified by tic in solvents A and B with known samples of pepstatin (R,: A, 0.56; B, 0.48) and tyrosine methyl ester (Rf: A, 0.15; B, 0.99). Compounds containing tyrosine were identified with uv light at 280 nm. Pepstatin-containing compounds were detected with a sulfuric acid-dichromate spray. The fractions containing pepstatin-L-tyrosine methyl ester (R;A, 0.90; B, 0.96) also contained unreacted pepstatin and required further purification. These fractions were pooled, evaporated to dryness, redissolved in l-2 ml of solvent B, and eluted by gravity flow from a silica gel column (1 x 25 cm) in the same solvent system, collecting fractions of 2 ml each. The content of each fraction was determined by tic as described above. Pepstatin-tyrosine methyl ester eluted first and was totally separated from unreacted pepstatin. The pepstatin-tyrosine methyl ester appeared homogeneous by tic. The fractions containing pepstatin-tyrosine methyl ester were pooled, evaporated to dryness in a tared vessel to calculate yield, and saved for iodination with lz51. In synthesizing pepstatin-L-iodotyrosine methyl ester, the appropriate fractions from the LH20 column in absolute methanol were pooled, dried, and redissolved in 50% methanol-water with heating. A trace quantity of pepstatin-L-[‘251]monoiodotyrosine methyl ester prepared by radioiodinating pepstatin-L-tyrosine methyl ester as described below was added, and the mixture was eluted by gravity flow from LH20 (2.5 x 38 cm) in 50% methanol-water. The column effluent was passed through a uv flow cell at 280 nm to detect the desired product. The uv absorbance at 203.5 nm of fractions exhibiting no absorbance at 280 nm
PEPSIN
INHIBITION
BY IODINATED
was read in an Hitachi 100-60 spectrophotometer to detect the peptide bonds of unreacted pepstatin, the identity of which was confirmed by tic. Radioactivity in each fraction was also quantified to determine the elution position of the radioiodinated pepstatin analog. Iodination of pepstatin-tyrosine deriuatives. Iodinations of pepstatin analogs were performed utilizing a modified procedure of Hunter and Greenvvood (6). lZ51, 1 mCi, (approximately 4 x lo-” mol) was added to 50 ~1 of 0.1 M phosphate, pH 7.4, and 100 ~1 of absolute methanol containing about 10mHmol of pepstatin-tyrosine methyl ester. The reaction was started by adding 25 ~1 of a 0.014 M solution of chloramine-T in water and mixing for 20 s. The reaction was stopped by the addition of 50 ~1 of a 0.042 M solution of sodium metabisulfite in water. The iodination mixture was purified on two successive LH20 columns. Chromatography on the first LH20 column (1 x 60 cm) in methanol separated the desired product from unreacted 12jI. Elution from the second LH20 column (1 x 22 cm) in 50% methanol-water separated iodinated from noniodinated pepstatin-L-tyrosine methyl ester and resulted in a product of high specific activity and purity. Each fraction was diluted up to 5 ml with absolute methanol, stored at -2o”C, and appeared to be stable for at least 2 months. Pepsin assay. Peptic activity was assayed using hemoglobin as a substrate. Bovine hemoglobin was dissolved in 0.05 M glycine-HCI, pH 2.0, and dialyzed overnight at 4°C against a 40-fold excess of the same buffer to reduce the blank. The final concentration of hemoglobin was then adjusted to 2%. Porcine pepsin was dissolved in 0.02 M citrate-phosphate, pH 4.5, immediately prior to use. The uv absorbance of both the undiluted pepsin stock solution and pepsin diluted with the identical amount of ethanol employed to add pepstatin compounds was measured and compared with a published value for the molar extinction coefficient (19). The concentration of pepsin was calculated from the optical density; and except for a dilution effect, the addition of ethanol did not change the absorbance. Pepsin and either pepstatin or pepstatinL-iodotyrosine methyl ester were mixed in the citratephosphate buffer at 4°C. One hundred microliters of a pepsin control solution or 100 ~1 of these pepsin-inhibitor stock solutions was then added to 5 ml of the hemoglobin solution which had been prewarmed to 37°C. The reaction was terminated at precisely 10 min by adding 5 ml of 10% trichloroacetic acid and immersing in ice. After centrifugation the absorbance of the supernatants at 280 nm was measured. Equilibrium dialysis. Equilibrium dialysis was performed to determine the dissociation constants of pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester. The reactants were initially mixed in 0.001 N HCI so that the final concentration of all pepstatin compounds was 1O-y M, whereas that of pepsin was varied from 0.31-2.5 x 10m9M. Pepstatin-L-
PEPSTATIN
159
[1251]monoiodotyrosine methyl ester was added as the trace, representing about 0.25% of the total pepstatin species present. Aliquots of 1 ml of each pepsin-inhibitor mixture were then added to individual cellulose dialysis sacs (0.25 in. diameter; permeable to 12,000 molecular weight), which were placed in individual polypropylene tubes containing 50 ml of 0.05 M citrate-phosphate, pH 5.0. Aliquots of 1 ml of each solution were also pipetted into five counting tubes to determine the average actual total counts (?‘) added to each dialysis sac and to calculate the specific activity of radioiodinated pepstatin for each inhibitor and each concentration of pepsin. After shaking for 336 h at 4”C, at which time equilibrium had been attained, the sac contents were weighed and counted. The observed radioactivity was expressed as counts per minute per milliliter of sac contents. Since the specific activity of the pepstatin-L-[‘Z”I]monoiodotyrosine methyl ester was known, the radioactivity could be converted to a molar concentration of all pepstatin species within the sac (i,,). The radioactivity of the fluid bathing the sac was calculated by subtracting the total observed radioactivity within the dialysis sac from the actual total counts (7’) and dividing by 30 to yield counts per minute per milliliter. This value was also converted to a molar concentration of pepstatin (i,,,,). The dissociation constant K,] could then be calculated using the equation of Johnson and Knowles (7) K,, = i,,,.,(e - Ai)/di, where e = enzyme concentration within the dialysis sac and Al = I,~ - i,,,,. The data were also analyzed using the linear regression of l/f’ on m/b, as suggested by Nimmo et ul. (16), where f‘ = i,,r,, rrl = Y and b = i,,. The slope of the line generated is the inverse of the dissociation constant, l/K,,. RESULTS
Synthesis of Pepstatin-Tyrosine Derivatives
Pepstatin was coupled to L-tyrosine methyl ester and L-iodotyrosine methyl ester, and each reaction mixture was purified initially on LHZO in methanol. The elution profile was similar with both tyrosine derivatives and that seen with iodotyrosine is depicted in Fig. 1. Three well-defined peaks were reproducibly observed, the first of which contained both the desired product and unreacted pepstatin. Pepstatin tyrosine methyl ester was further purified to homogeneity on a silica gel column, and the observed yield ranged from 31-‘78% of that predicted. Pepstatin-L-iodotyrosine methyl ester rapidly became discolored in solvent system B utilized for silica gel chromatography; and therefore, this
160
WORKMAN
0
30
40 FRACTION
50 NUMBER
60
AND BURKITT
70
FIG. 1. Purification of pepstatin-L-iodotyrosine methyl ester on LH20 in methanol. Pepstatin was coupled to L-iodotyrosine methyl ester and purified on LH20 (1 x 110 cm) in methanol, collecting fractions of 1.6 ml. Peak I contained pepstatin-L-iodotyrosine methyl ester and unreacted pepstatin, peak II N-p(P-carbomethoxy, p-amino ethyl) iodophenyl symmetrical dicyclohexylurea (the condensation product of iodotyrosine methyl ester and dicyclohexylcarbodiimide), and III L-iodotyrosine methyl ester and dicyclohexylurea.
compound was purified on LH20 in aqueous methanol instead. A typical chromatographic run is illustrated in Fig. 2. The elution profile of pepstatin-iodotyrosine methyl ester (peak III), detected by absorbance at 280 nm, was exactly superimposable upon
FIG. 2. Purification of pepstatin-L-iodotyrosine methyl ester on LH20 in aqueous methanol. Peak I from an LH20 column similar to that in Fig. 1 was mixed with a trace quantity of pepstatin-L-[1251]iodotyrosine methyl ester in 50% methanol and chromatographed on LHZO (2.5 x 38 cm) in the same solvent, collecting fractions of 7 ml. Peak I represented unreacted pepstatin, II pepstatin-L-tyrosine methyl ester, and III pepstatin-L-iodotyrosine methyl ester (containing both stable iodine and 9).
that of pepstatin-[1251]monoiodotyrosine methyl ester which had been prepared in a different manner by iodinating pepstatin tyrosine methyl ester with chloramine-T. A small amount of pepstatin-L-tyrosine methyl ester (peak II) probably was formed by deiodination of the iodo derivative. The absorbance of pepstatin in peak I ~~~~~~~ = 17051) appeared much greater than that of the iodotyrosine derivative (eZS,,= 2380) due to the difference in the molar extinction coefficients. Beginning with 30 mg of pepstatin the final yield of the iodo derivative after all chromatographic steps was 38%. The actual peak of maximum uv absorbance occurred at 286 nm with E = 2616. Iodination Ester
of Pepstatin-Tyrosine
Methyl
Using an LH20 column in methanol (Fig. 3) to purify the radioiodinated pepstatin, the product eluted at the void volume, had a specific activity ranging from 30-130 Ci/ mmol and was well separated from unreacted lz51. If the iodination mixture was chromatographed initially on LH20 in 50% methanol-water (Fig. 4), the elution pattern was totally different, the unreacted lz51 (peak III) appearing well before the radio-
FRACTION
NUMBER
FIG. 3. Purification of pepstatin-L-[1251]iodotyrosine methyl ester on LH20 in methanol. Following iodination of pepstatin-L-tyrosine methyl ester the reaction mixture was eluted from LH20 (1 x 60 cm) in methanol, collecting fractions of 1.3 ml. The first peak represented pepstatin-L-[‘251]iodotyrosine methyl ester and the second unreacted lz51.
PEPSIN INHIBITION
BY IODINATED
PEPSTATIN
161
0.015 t w 0.010 ks 2 5 0.005 :
0 FRACTION
NUMBER
FIG. 4. Purification of pepstatin-L-[*251]iodotyrosine methyl ester on LH20 in 50% methanol-water. The iodination mixture was chromatographed on LH20 (1 x 22 cm) in 50% methanol-water, collecting fractions of 0.76 ml. Following this procedure, the same column was standardized by eluting individually blue dextran to mark the V,, a mixture of sodium metabisulhte and chloramine-T, sodium iodide, p-toluene sulfonamide (the reduction product of chloramine-T), and each of the other reactants of the iodination reaction. The identity of the peaks detected was assigned as follows: , IV, pepstatin-L-tyrosine methyl ester; V, p-toluene I, sodium metabisulfite; II, chloramine-T; III, lz51. sulfonamide; and VI, pepstatin-L-[1*51]iodotyrosine methyl ester.
iodinated pepstatin (peak VI). Of major importance was the finding that unreacted pepstatin-L-tyrosine methyl ester (peak IV) eluted before and was completely separated from its radioiodinated analog. Therefore, the specific activity of the product was markedly enriched. Peak V, representing p-toluene sulfonamide or the reduction product of chloramine-T, eluted at the beginning of the radioiodinated pepstatin peak. This material could be completely eliminated by eluting the iodination mixture first on LHZO in absolute methanol and eluting the product peak appearing at the V, on the second LH20 column in 50% methanol-water. Peak VI then eluted in the expected position, but there was no demonstrable uv absorbance due to the elimination of p-toluene sulfonamide and to the infinitesimal mass of the desired product. TIC and radioautography of the product in solvent systems B, C, and D demonstrated a single spot migrating with the same R, (B, 0.920; C, 0.966; D, 0.982) of nonradioactive pepstatin-iodotyrosine methyl ester and containing 9597.4% of the radioactivity of each tic plate.
min incubation was, therefore, equivalent to an initial reaction velocity 2). Since pepstatin is a tight-binding inhibitor of pepsin, a plot of u against inhibitor concentration can be used to determine the concentration of enzyme catalytic sites (15) assuming an equimolar reaction between pepsin and pepstatin or to determine the stoichiometry of the enzyme-inhibitor complex if the reaction is not equimolar. At v = 0, 100% inhibition is achieved; and the intercept on the abscissa represents the concentration of inhibitor combined with enzyme at this point. As shown in Fig. 5, with 1.22 x lop7 M pepsin this value was 1.15 x 10e7M for pepstatin and 1.095 x 10e7M for pepstatin-Liodotyrosine methyl ester. These results indicated that both pepstatin species were almost equally effective inhibitors of peptic activity against hemoglobin at pH 2.0 and that the ratio of enzyme/inhibitor at 100% inhibition was 1.06 for pepstatin and 1.12 for the iodinated pepstatin. Equilibrium
Dialysis
The results of the equilibrium dialysis experiments obtained with the equation of Pepsin Activity Assay Johnson and Knowles (7) are summarized in It was established that the reaction of Table I. The KD obtained with pepstatin pepsin with hemoglobin under the condi- was 3.49-fold greater than that with the iotions specified was linear with time; and the dinated derivative (P < 0.004), suggesting absorbance of the supernatants after a lo- that the latter combined somewhat more
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WORKMAN AND BURKITT
INHIBITOR
CONCENTRATION
X lOa MIL
FIG. 5. Inhibition of pepsin by pepstatin and pepstatin-L-iodotyrosine methyl ester. The residual proteolytic activity of a 1.22 x 10m7M solution of pepsin was assayed with bovine hemoglobin as substrate. For each inhibitor the correlation coefficient, slope, and intercept of the data were calculated by the method of least squares: pepstatin (O), r = -0.99418, m = -0.10073, x: intercept = 1.15 X 10-’ M; pepstatinL-iodotyrosine methyl ester (A), r = -0.99260, m = -0.10016, x intercept = 1.095 x 10m7M.
strongly with pepsin at pH 5.0 than did pepstatin itself. The data were also analyzed by plotting lliouT vs e/i,, (Fig. 6), a linear regression which according to Nimmo et al. (16) tends to minimize random errors. This analysis tended to emphasize the difference between the dissociation constants which had been determined with the equation of Johnson and Knowles (‘7). From these results it definitely appeared that pepstatinL-iodotyrosine methyl ester was at least as good an inhibitor of pepsin as is pepstatin itself and may even have bound more tightly by a factor of 3-14.
FIG. 6. Determination of the K,, for pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester by equilibrium dialysis. The results of equilibrium dialysis are plotted as l/i,,r,r against e/i,, for pepsin-pepstatin (0) and pepsin-pepstatin-L-iodotyrosine methyl ester (A). The data were analyzed by the method of least squares. The K,, for each complex is equivalent to the inverse of the slope ?rtor 1.38 x 10. ‘” M for the former and 9.83 x 1Om’2M for the latter enzyme-inhibitor complex.
DISCUSSION
The past decade has witnessed the important development of specific inhibitors of the acid proteases. Of all the inhibitors described pepstatin is the most potent by several orders of magnitude, the K, or Ki having been reported to lie within the range of lo-‘to lo-” M for pepsin (9- 12,21), renin (ll), and cathepsin D (8). The unique inhibitory capacity of pepstatin can probably be
TABLE I DETERMINATION OF K, FOR PEPSIN-PEPSTATIN AND PEPSIN-PEPSTATIN-L-IODOTYROSINE METHYL ESTER BY EQUILIBRIUM DIALYSIS
Pepstatin-L-iodotyrosine methyl ester (1.04 X It9 M)
Pepstatin (1.02 X lo-' M)
Inhibitor species Pepsin concentration x 1OmyM
2.5
1.2
0.62
0.31
2.5
1.2
0.62
0.31
K,
6.68
4.10
3.62
3.86
0.93
1.19
2.23
0.89
x
lo-”
M'"
Mean f SD
4.57 5 1.42
n Calculated using the equation of Johnson and Knowles K, = i,,&
1.31 k 0.63 - Ai)/Ai.
PEPSIN
INHIBITION
BY IODINATED
largely attributed to the two statine (4amino, 3-hydroxyl, 6-methyl heptanoic acid) residues within the molecule (12, 18), and pepstatin seems to inhibit the acid proteases at a ratio of enzyme/inhibitor ranging from 1.0-2.0 depending upon the pH (10, 12). Due to the tremendous inhibitory potential of pepstatin many applications for this compound can be envisioned in the study of the acid proteases. Consequently, it would be very useful to have a radiolabeled pepstatin which would be obtainable at an accurately known specific activity. The major underlying assumption would be that in radiolabeling the pepstatin molecule, the inhibitory characteristics of native pepstatin towards the acid proteases would be unchanged. In contrast to the relatively low specific activity pepstatin derivatives which have been prepared in the past (8-10, 14), this report describes the synthesis of an iodinated pepstatin of very high specific activity. Due to the fact that iodine-containing compounds are retarded on Sephadex gels, this property has been exploited to obtain iodinated pepstatins (with both stable and radioactive isotopes of iodine) of very high purity and/or specific activity. The identity and purity of the radioiodinated pepstatin was confirmed by its coelution from LH20 in aqueous methanol and from its identical migration in three tic systems with the nonradioactive pepstatin-L-iodotyrosine methyl ester. Quantification of the radioactivity seen in radioautographs of the tic plates revealed that virtually all of it was localized to the pepstatin-L-[1251]iodotyrosine methyl ester. The important requirement that the inhibitory properties of radiolabeled pepstatin toward the acid proteases remain unchanged has been demonstrated with the iodinated derivatives. Peptic activity was assayed with hemoglobin as a substrate to compare the behavior of pepstatin and pepstatin-L-iodotyrosine methyl ester with each other. Previous modifications of pepstatin, including esterification with methyl (8, 21), ethyl (21), and p-bromophenacyl groups (21), conversion to the amide (21), and coupling to diaminoethane (8) and glytine (8), altered the activity of pepstatin toward pepsin and cathepsin D either not
PEPSTATIN
163
at all or only slightly. In agreement with these findings, both pepstatin and pepstatin-L-iodotyrosine methyl ester generated almost identical regressions of v (or absorbance) on inhibitor concentration (Fig. 5). In addition, the stoichiometry of the reaction could also be estimated by extrapolating the inhibition curves to 100% inhibition or 0% enzyme activity at pH 2.0. The ratios of enzyme/inhibitor were 1.06 for pepstatin and 1.12 for pepstatin+iodotyrosine methyl ester. Kunimoto et al. (lo), using the synthetic peptide substrates N-acetyl-L-phenylalanyl+diiodotyrosine at pH 2.0 and Phe-Gly-His-Phe(NO,)-PheAla-Phe-O-Me at ph 4.0, concluded that the reaction of pepstatin with pepsin was equimolar, and used this information to titrate pepsin activity against known amounts of pepstatin to calculate enzyme concentration. Marciniszyn et aZ. (12) came to a somewhat different conclusion since they demonstrated a variation in the stoichiometry of the reaction with pH. These authors postulated that the stoichiometry may vary from 1 to 2, especially at pH 2.0 where each statine residue may combine with an enzyme molecule. Using intact pepstatin and hemoglobin as substrate, the ratio of enzyme to pepstatin was 1.96 at pH 2.1, 1.33 at pH 3.1, and 1.13 at pH 3.65. Using a pepstatin fragment N-acetyl-valyl-statylalanyl-statine and N-acetyl-L-phenylalanylL-diiodotyrosine as substrate, the enzyme/ inhibitor ratio varied at random from 1.61 to 1.89 over a pH range of 2.1 to 4.5. In the experiments reported here peptic activity was assayed at pH 2.0. Our results tend to support Kunimoto’s conclusions that the pepsin-pepstatin reaction is equimolar and that any deviation from this ratio is due to inactive enzyme. Our findings also demonstrate that the inhibitory capacity of pepstatin was essentially unchanged by the chemical modification at its carboxyl terminus. An accurate determination of either the Ki by kinetic methods or the K, by equilibrium dialysis would provide the most precise estimate of the strength of the pepsinpepstatin interaction. The interpretation of kinetic studies is limited, however, by the fact that the traditional substrates for pep-
164
WORKMAN
AND BURKITT
sin technically cannot be used within the concentration range of the Ki. The availability of high specific activity radioiodinated pepstatin which could be diluted to trace quantities at precisely calculated specific activities with nonlabeled pepstatin species permitted the performance of equilibrium dialysis at a much lower concentration range of pepsin and pepstatin. Both the equation of Johnson and Knowles (7) and the linear regression of l/ZoUT vs e/i,, of Nimmo et al. (16) were used to calculate the K,. The values obtained indicated that under the conditions specified, pepstatin-Liodotyrosine methyl ester actually appeared to form a more stable complex with pepsin than did native pepstatin. These values agreed very well with the Ki of 9.7 x lo-” M for the pepsin-pepstatin complex obtained by Kunimoto et al. (10) using the synthetic substrate Phe-Gly-His-Phe(NO,)-Phe-Ala-Phe-O-Me at pH 4.0, but were considerably lower than the values of 1.2-1.7 x 1O-gM determined by McKown et al. (11) using an ‘251-labeled synthetic polymeric substrate at pH 5.5 and 1.1 x 10egM found by Marciniszyn et al. (12) using hemoglobin as substrate at pH 2.1 and assuming that two pepsin molecules combine with each pepstatin molecule at this pH. The equilibrium dialysis studies provide further evidence that the radiolabeled pepstatin behaves towards pepsin essentially in the same manner as does native, unmodified pepstatin. This finding suggests that radioiodinated pepstatin might be useful as an affinity label in the study of the entire class of acid proteases. ACKNOWLEDGMENTS We are greatly indebted to Dr. Tadashi Inagami for his many helpful suggestions and review of the manuscript. REFERENCES 1. AOYAGI, T., MORISHIMA, H., NISHIZAWA, R., KUNIMOTO, S., TAKEUCHI, T., UMEZAWA, H., AND IZEKAWA, H. (1972) J. Antibiot. 25. 689-694.
2. BARRETT, A. J., AND DINGLE, J. T. (1972) Biothem. J. 127, 439-441. 3. FERGUSON, J. B., ANDREWS, J. R., VOYNICK, I. M., AND FRUTON, J. S. (1973)J. Biol. Chem. 248, 6701-6708. 4. GROSS, F., LAZAR, J., AND ORTH, H. (1972) Sciewe 175, 656. 5. GUYENE, T. T., DEVAUX, C., MENARD, J., AND CORVOL, P. (1976) J. Cl&. Endocrino~. 43, 1301-1306. 6. HUNTER, W. M., AND GREENWOOD, F. C. (1962) Nature (London) 194, 495-496. 7. JOHNSON, C. H., AND KNOWLES, J. R. (1966) Bzothem. J. 101, 56-62. 8. KNIGHT, C. G., AND BARRETT, A. J. (1976) Bio&em. J. 155, 117-125. 9. KUNIMOTO, S., AOYAGI, T., MORISHIMA, H., TAKEUCHI, T., ANDUMEZAWA, H. (1972)J. Antibiot. 25, 251-255. 10. KUNIMOTO, S., AOYAGI, T., NISHIZAWA, R., KOMAI, T., TAKEUCHI, T., AND UMEZAWA, H. (1974) J. Antibiot. 27, 413-418. 11. MCKOWN, M. M., WORKMAN, R. J., ANDGREGERMAN, R. I. (1974) J. Biol. Chem. 249, 77707774. 12. MARCINISZYN, J., HARTSUCK, J. A., AND TANG, J. (1976) J. Biol. Chem. 251, 7088-7094. 13. MILLER, R. P., POPER, C. J., WILSON, C. W., AND DEVITO, E. (1972) Biochem. Pharmacol. 21, 2941-2944. 14. MORISHIMA, H., SAWA, T., TAKITA, T., AOYAGI, T., TAKEUCHI, T., AND UMEZAWA, H. (1974) J. Antibiot. 27, 267-273. 15. MORRISON, J. F. (1969) Biochim. Biophys. Acta. 185, 269-286. 16. NIMMO, I. A., ATKINS, G. L., STRANGE, R. C., AND PERCY-R• BB, I. W. (1977) Biochem. .J. 165, 107- 110. 17. ORTH, H., HACKENTHAL, E., LAZAR, J., MIKSCHE, U., AND GROSS, F. (1974) Circ. Res. 35,52-55. 18. RICH, D. H., SUN, E., AND SINGH, J. (1977) Biothem. Biophys. Res. Commun. 74, 762-767. 19. RYLE, A. P. (1970) in Methods of Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 316-336, Academic Press, New York. 20. SUBRAMANIAN, E., SWAN, I. D. A., Lru, M., DAVIES, D. R., JENKINS, J. A., TICKLE, I. J., AND BLUNDELL, T. L. (1977) Proc. Nat. Acad. Sci. UISA 74, 556-559. 21. UMEZAWA, H., AOYAGI, T., MORISHIMA, H., MATSUZAKI, M., HAMADA, M., ANDTAKEUCHI, T. (1970) J. Antibiot. 23, 259-262. 22. WOESSNER, J. F. (1972) Biochem. Biophys. Res. Commun. 47, 965-970.
Pepsin inhibition
by a High Specific Activity Derivative of Pepstatinl
ROBERT JAY WORKMAN’ Veteraw
Administration Received
Hospital, September
AND
Radioiodinated
DONNA W. BURKITT
1310 24th Avenue South, Nashville,
Tennessee SROd
18, 1978; revised October 11. 1978
High specific activity radioiodinated derivatives of pepstatin which retain the inhibitory properties of the parent compound toward pepsin have been prepared. Pepstatin was first coupled to L-tyrosine methyl ester or L-3-iodotyrosine methyl ester. Pepstatin-Ltyrosine methyl ester was iodinated with lz51 and chloramine-T and purified to homogeneity on LH20 columns. Nonradioactive pepstatin-L-iodotyrosine methyl ester and pepstatinL-[1251]monoiodotyrosine methyl ester coeluted in gel filtration column chromatography. Using thin layer chromatography and radioautography a mixture of both these compounds also migrated as a single spot. On the basis of these criteria the radioiodinated pepstatin was judged to be virtually pure and have a specific activity of 2.16 Ci/pmol. Using the digestion of hemoglobin to assay peptic activity at pH 2.0, both pepstatin and pepstatinL-iodotyrosine methyl ester demonstrated similar inhibition of pepsin, indicating that the addition of the iodotyrosine methyl ester group to the carboxyl terminus of pepstatin did not alter the inhibitory properties of the parent compound. By means of equilibrium dialysis at pH 5.0 in which pepstatin-L-[‘2jI]monoiodotyrosine methyl ester was used in trace quantities, the dissociation constants K, of pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester were determined and found to be 4.57 x 10-l’ _f 1.42 M and 1.31 x 10-l’ ? 0.63 M respectively. The closeness of these values to each other provides additional quantitative evidence that pepstatin-L-iodotyrosine methyl ester reacts with pepsin in a manner similar to that observed with native, unmodified pepstatin and suggests that pepstatin-L-[““Ilmonoiodotyrosine methyl ester may be used as an affinity label of acid proteases under the same circumstances in which native pepstatin is known to react with this class of enzymes.
The most potent known inhibitor of the so-called acid protease class of enzymes is the bacterial fermentation product pepstatin (isovaleryl-L-valyl-L-valyl-4-amino3-hydroxy-6-methyl heptanoyl-L-alanyl-4amino-3-hydroxyl-6-methyl heptanoic acid). Due to its potency this substance has found numerous applications in studying the biochemistry of the enzymes which it inhibits, most notably pepsin (1, 9-11, 20, 21), cathepsin D (1, 2, 3, 8, 22), renin (1, 4, 5, 11, 13, 17), and fungal acid proteases (20, 21). Several of these studies have been facilitated by utilizing radioactive derivatives of pepstatin labeled with “C or 3H. Kunimoto ’ This investigation was supported by the Medical Research Service of the Veterans Administration, * To whom correspondence should be addressed.
(9, 10) studied the binding of pepstatin to pepsin using tritiated pepstatin of specific activity 68 ~Cil~mol prepared by exposing pepstatin to tritium gas. 14C-Labeled pepstatins with a maximum specific activity of 164 &X/mmol were obtained by Morishima et al. (14) by incubating cultures of Streptomyces testaceus with 14C-labeled amino acids and isolating the pepstatins released into the culture broth. Knight and Barrett (8) coupled pepstatin to [3H]glycine to obtain a product with a specific activity of 31 &i/pmol which was at least 50% impure since it contained both uncoupled pepstatin and some other unidentified contaminant which did not bind to cathepsin D. These radiolabeled pepstatins possessed low specific activity and in the last report the product was impure. The present study describes et al.
157
0003.9861/79/050157-08$02.00/0 Copyright G 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
158
WORKMAN AND BURKITT
the preparation of a radioiodinated pepstatin derivative of very high specific activity and purity which retains the inhibitory properties of the parent compound. The applicability of such a compound is illustrated by using the radioiodinated pepstatin in trace quantities at an accurately known specific activity to determine the dissociation constants of pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester. EXPERIMENTAL
PROCEDURE
Materials Pepstatin A was a gift from H. Umezawa (Tokyo, Japan). Porcine pepsin, twice crystallized (specific activity 4500 U/mg of protein; lot SSC-SOSO), L-tyrosine methyl ester, and L-3-iodotyrosine were from Sigma Chemical Company, St. Louis, Missouri. Dicyclohexylcarbodiimide, chloramine-T, cellulose tic sheets with fluorescent indicator and no-screen medical X-ray film NSZT were from the Eastman Kodak Company, Rochester, New York. Bovine hemoglobin, twice crystallized, was obtained from Miles Laboratories, Inc., Kankakee, Illinois. Spectral grade solvents were from J. T. Baker, Phillipsburg, New Jersey. Sulfuric acid-dichromate spray and 3 N methanol&HCl were from Supelco, Inc., Bellefonte, Pennsylvania. LH20 was obtained from Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey. Silicar CC-4 was from Mallinckrodt, Inc., St. Louis, Missouri, Carrier-free high concentration (17 Ciimg) sodium [1251]iodidewas obtained from New England Nuclear, Boston, Massachusetts. Merck precoated tic silica gel 60 F254 sheets of 0.24 mm-thickness with fluorescent indicator on aluminum backing were obtained through Brinkmann Instruments, Inc., Des Plaines, 111. Other reagent grade chemicals were obtained from various commercial sources.
Methods Thin layer chromatography (tic). The following tic solvent systems3 were used: (A) chloroform:methanol:acetic acid, 86:9:5; (B) chloroform:methanol: ammonia, 75:24:1; (C) ethyl acetate:pyridine:acetic acid:water, 35:35:7:14; (D) isopropanol:ethyl acetate: 6 N ammonia:acetone, 35:30:25:20. Solvents A and B were used with silica gel tic plates on aluminum 3 Abbreviations used: Solvent A, chloroform:methanobacetic acid, 86:9:5; solvent B, chloroform:methanol: ammonia, 75:24:1; solvent C, ethyl acet.ate:pyridine: acetic acid:water, 35:35:7:14; solvent D, isopropanol: ethyl acetate:6 N ammonia:acetone, 35:30:25:20; E, total counts; e, enzyme concentration.
backing. Solvents C and D were used with cellulose tic plates on plastic. After performing tic of radioiodinated compounds, the tic plate was pressed against Kodak no-screen X-ray film to produce a radioautograph. The coating of appropriate tic plates was scraped off and counted in a well scintillation counter (Searle Model 1197, efficiency 78%) to quantitate the actual radioactivity in each spot seen on radioautography. Coupling of’ pepstatin to tyrosine esters. L-3-Iodotyrosine was esterified with 3 N methanolic-HCl and purified on LH20 (1 x 22 cm) in 50% methanolwater. Purity of the product was confirmed by tic in solvent system B. L-tyrosine methyl ester (known) and L-iodotyrosine methyl ester had an R, of 0.989, whereas the free acid had an R, of 0.473. Pepstatin, tyrosine methyl ester, and dicyclohexylcarbodiimide in molar proportions of approximately 1:4:4 were suspended in 8-10 ml of dichloromethane and stirred at room temperature for 96 h. The solvent was removed by evaporation, and the residue was redissolved in methanol and eluted from an LH20 column (1 x 110 cm) in methanol by gravity flow through a uv flow cell (ISCO Model UA5 with Type 6 optical unit) at 280 nm. Products were identified by tic in solvents A and B with known samples of pepstatin (R,: A, 0.56; B, 0.48) and tyrosine methyl ester (Rf: A, 0.15; B, 0.99). Compounds containing tyrosine were identified with uv light at 280 nm. Pepstatin-containing compounds were detected with a sulfuric acid-dichromate spray. The fractions containing pepstatin-L-tyrosine methyl ester (R;A, 0.90; B, 0.96) also contained unreacted pepstatin and required further purification. These fractions were pooled, evaporated to dryness, redissolved in l-2 ml of solvent B, and eluted by gravity flow from a silica gel column (1 x 25 cm) in the same solvent system, collecting fractions of 2 ml each. The content of each fraction was determined by tic as described above. Pepstatin-tyrosine methyl ester eluted first and was totally separated from unreacted pepstatin. The pepstatin-tyrosine methyl ester appeared homogeneous by tic. The fractions containing pepstatin-tyrosine methyl ester were pooled, evaporated to dryness in a tared vessel to calculate yield, and saved for iodination with lz51. In synthesizing pepstatin-L-iodotyrosine methyl ester, the appropriate fractions from the LH20 column in absolute methanol were pooled, dried, and redissolved in 50% methanol-water with heating. A trace quantity of pepstatin-L-[‘251]monoiodotyrosine methyl ester prepared by radioiodinating pepstatin-L-tyrosine methyl ester as described below was added, and the mixture was eluted by gravity flow from LH20 (2.5 x 38 cm) in 50% methanol-water. The column effluent was passed through a uv flow cell at 280 nm to detect the desired product. The uv absorbance at 203.5 nm of fractions exhibiting no absorbance at 280 nm
PEPSIN
INHIBITION
BY IODINATED
was read in an Hitachi 100-60 spectrophotometer to detect the peptide bonds of unreacted pepstatin, the identity of which was confirmed by tic. Radioactivity in each fraction was also quantified to determine the elution position of the radioiodinated pepstatin analog. Iodination of pepstatin-tyrosine deriuatives. Iodinations of pepstatin analogs were performed utilizing a modified procedure of Hunter and Greenvvood (6). lZ51, 1 mCi, (approximately 4 x lo-” mol) was added to 50 ~1 of 0.1 M phosphate, pH 7.4, and 100 ~1 of absolute methanol containing about 10mHmol of pepstatin-tyrosine methyl ester. The reaction was started by adding 25 ~1 of a 0.014 M solution of chloramine-T in water and mixing for 20 s. The reaction was stopped by the addition of 50 ~1 of a 0.042 M solution of sodium metabisulfite in water. The iodination mixture was purified on two successive LH20 columns. Chromatography on the first LH20 column (1 x 60 cm) in methanol separated the desired product from unreacted 12jI. Elution from the second LH20 column (1 x 22 cm) in 50% methanol-water separated iodinated from noniodinated pepstatin-L-tyrosine methyl ester and resulted in a product of high specific activity and purity. Each fraction was diluted up to 5 ml with absolute methanol, stored at -2o”C, and appeared to be stable for at least 2 months. Pepsin assay. Peptic activity was assayed using hemoglobin as a substrate. Bovine hemoglobin was dissolved in 0.05 M glycine-HCI, pH 2.0, and dialyzed overnight at 4°C against a 40-fold excess of the same buffer to reduce the blank. The final concentration of hemoglobin was then adjusted to 2%. Porcine pepsin was dissolved in 0.02 M citrate-phosphate, pH 4.5, immediately prior to use. The uv absorbance of both the undiluted pepsin stock solution and pepsin diluted with the identical amount of ethanol employed to add pepstatin compounds was measured and compared with a published value for the molar extinction coefficient (19). The concentration of pepsin was calculated from the optical density; and except for a dilution effect, the addition of ethanol did not change the absorbance. Pepsin and either pepstatin or pepstatinL-iodotyrosine methyl ester were mixed in the citratephosphate buffer at 4°C. One hundred microliters of a pepsin control solution or 100 ~1 of these pepsin-inhibitor stock solutions was then added to 5 ml of the hemoglobin solution which had been prewarmed to 37°C. The reaction was terminated at precisely 10 min by adding 5 ml of 10% trichloroacetic acid and immersing in ice. After centrifugation the absorbance of the supernatants at 280 nm was measured. Equilibrium dialysis. Equilibrium dialysis was performed to determine the dissociation constants of pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester. The reactants were initially mixed in 0.001 N HCI so that the final concentration of all pepstatin compounds was 1O-y M, whereas that of pepsin was varied from 0.31-2.5 x 10m9M. Pepstatin-L-
PEPSTATIN
159
[1251]monoiodotyrosine methyl ester was added as the trace, representing about 0.25% of the total pepstatin species present. Aliquots of 1 ml of each pepsin-inhibitor mixture were then added to individual cellulose dialysis sacs (0.25 in. diameter; permeable to 12,000 molecular weight), which were placed in individual polypropylene tubes containing 50 ml of 0.05 M citrate-phosphate, pH 5.0. Aliquots of 1 ml of each solution were also pipetted into five counting tubes to determine the average actual total counts (?‘) added to each dialysis sac and to calculate the specific activity of radioiodinated pepstatin for each inhibitor and each concentration of pepsin. After shaking for 336 h at 4”C, at which time equilibrium had been attained, the sac contents were weighed and counted. The observed radioactivity was expressed as counts per minute per milliliter of sac contents. Since the specific activity of the pepstatin-L-[‘Z”I]monoiodotyrosine methyl ester was known, the radioactivity could be converted to a molar concentration of all pepstatin species within the sac (i,,). The radioactivity of the fluid bathing the sac was calculated by subtracting the total observed radioactivity within the dialysis sac from the actual total counts (7’) and dividing by 30 to yield counts per minute per milliliter. This value was also converted to a molar concentration of pepstatin (i,,,,). The dissociation constant K,] could then be calculated using the equation of Johnson and Knowles (7) K,, = i,,,.,(e - Ai)/di, where e = enzyme concentration within the dialysis sac and Al = I,~ - i,,,,. The data were also analyzed using the linear regression of l/f’ on m/b, as suggested by Nimmo et ul. (16), where f‘ = i,,r,, rrl = Y and b = i,,. The slope of the line generated is the inverse of the dissociation constant, l/K,,. RESULTS
Synthesis of Pepstatin-Tyrosine Derivatives
Pepstatin was coupled to L-tyrosine methyl ester and L-iodotyrosine methyl ester, and each reaction mixture was purified initially on LHZO in methanol. The elution profile was similar with both tyrosine derivatives and that seen with iodotyrosine is depicted in Fig. 1. Three well-defined peaks were reproducibly observed, the first of which contained both the desired product and unreacted pepstatin. Pepstatin tyrosine methyl ester was further purified to homogeneity on a silica gel column, and the observed yield ranged from 31-‘78% of that predicted. Pepstatin-L-iodotyrosine methyl ester rapidly became discolored in solvent system B utilized for silica gel chromatography; and therefore, this
160
WORKMAN
0
30
40 FRACTION
50 NUMBER
60
AND BURKITT
70
FIG. 1. Purification of pepstatin-L-iodotyrosine methyl ester on LH20 in methanol. Pepstatin was coupled to L-iodotyrosine methyl ester and purified on LH20 (1 x 110 cm) in methanol, collecting fractions of 1.6 ml. Peak I contained pepstatin-L-iodotyrosine methyl ester and unreacted pepstatin, peak II N-p(P-carbomethoxy, p-amino ethyl) iodophenyl symmetrical dicyclohexylurea (the condensation product of iodotyrosine methyl ester and dicyclohexylcarbodiimide), and III L-iodotyrosine methyl ester and dicyclohexylurea.
compound was purified on LH20 in aqueous methanol instead. A typical chromatographic run is illustrated in Fig. 2. The elution profile of pepstatin-iodotyrosine methyl ester (peak III), detected by absorbance at 280 nm, was exactly superimposable upon
FIG. 2. Purification of pepstatin-L-iodotyrosine methyl ester on LH20 in aqueous methanol. Peak I from an LH20 column similar to that in Fig. 1 was mixed with a trace quantity of pepstatin-L-[1251]iodotyrosine methyl ester in 50% methanol and chromatographed on LHZO (2.5 x 38 cm) in the same solvent, collecting fractions of 7 ml. Peak I represented unreacted pepstatin, II pepstatin-L-tyrosine methyl ester, and III pepstatin-L-iodotyrosine methyl ester (containing both stable iodine and 9).
that of pepstatin-[1251]monoiodotyrosine methyl ester which had been prepared in a different manner by iodinating pepstatin tyrosine methyl ester with chloramine-T. A small amount of pepstatin-L-tyrosine methyl ester (peak II) probably was formed by deiodination of the iodo derivative. The absorbance of pepstatin in peak I ~~~~~~~ = 17051) appeared much greater than that of the iodotyrosine derivative (eZS,,= 2380) due to the difference in the molar extinction coefficients. Beginning with 30 mg of pepstatin the final yield of the iodo derivative after all chromatographic steps was 38%. The actual peak of maximum uv absorbance occurred at 286 nm with E = 2616. Iodination Ester
of Pepstatin-Tyrosine
Methyl
Using an LH20 column in methanol (Fig. 3) to purify the radioiodinated pepstatin, the product eluted at the void volume, had a specific activity ranging from 30-130 Ci/ mmol and was well separated from unreacted lz51. If the iodination mixture was chromatographed initially on LH20 in 50% methanol-water (Fig. 4), the elution pattern was totally different, the unreacted lz51 (peak III) appearing well before the radio-
FRACTION
NUMBER
FIG. 3. Purification of pepstatin-L-[1251]iodotyrosine methyl ester on LH20 in methanol. Following iodination of pepstatin-L-tyrosine methyl ester the reaction mixture was eluted from LH20 (1 x 60 cm) in methanol, collecting fractions of 1.3 ml. The first peak represented pepstatin-L-[‘251]iodotyrosine methyl ester and the second unreacted lz51.
PEPSIN INHIBITION
BY IODINATED
PEPSTATIN
161
0.015 t w 0.010 ks 2 5 0.005 :
0 FRACTION
NUMBER
FIG. 4. Purification of pepstatin-L-[*251]iodotyrosine methyl ester on LH20 in 50% methanol-water. The iodination mixture was chromatographed on LH20 (1 x 22 cm) in 50% methanol-water, collecting fractions of 0.76 ml. Following this procedure, the same column was standardized by eluting individually blue dextran to mark the V,, a mixture of sodium metabisulhte and chloramine-T, sodium iodide, p-toluene sulfonamide (the reduction product of chloramine-T), and each of the other reactants of the iodination reaction. The identity of the peaks detected was assigned as follows: , IV, pepstatin-L-tyrosine methyl ester; V, p-toluene I, sodium metabisulfite; II, chloramine-T; III, lz51. sulfonamide; and VI, pepstatin-L-[1*51]iodotyrosine methyl ester.
iodinated pepstatin (peak VI). Of major importance was the finding that unreacted pepstatin-L-tyrosine methyl ester (peak IV) eluted before and was completely separated from its radioiodinated analog. Therefore, the specific activity of the product was markedly enriched. Peak V, representing p-toluene sulfonamide or the reduction product of chloramine-T, eluted at the beginning of the radioiodinated pepstatin peak. This material could be completely eliminated by eluting the iodination mixture first on LHZO in absolute methanol and eluting the product peak appearing at the V, on the second LH20 column in 50% methanol-water. Peak VI then eluted in the expected position, but there was no demonstrable uv absorbance due to the elimination of p-toluene sulfonamide and to the infinitesimal mass of the desired product. TIC and radioautography of the product in solvent systems B, C, and D demonstrated a single spot migrating with the same R, (B, 0.920; C, 0.966; D, 0.982) of nonradioactive pepstatin-iodotyrosine methyl ester and containing 9597.4% of the radioactivity of each tic plate.
min incubation was, therefore, equivalent to an initial reaction velocity 2). Since pepstatin is a tight-binding inhibitor of pepsin, a plot of u against inhibitor concentration can be used to determine the concentration of enzyme catalytic sites (15) assuming an equimolar reaction between pepsin and pepstatin or to determine the stoichiometry of the enzyme-inhibitor complex if the reaction is not equimolar. At v = 0, 100% inhibition is achieved; and the intercept on the abscissa represents the concentration of inhibitor combined with enzyme at this point. As shown in Fig. 5, with 1.22 x lop7 M pepsin this value was 1.15 x 10e7M for pepstatin and 1.095 x 10e7M for pepstatin-Liodotyrosine methyl ester. These results indicated that both pepstatin species were almost equally effective inhibitors of peptic activity against hemoglobin at pH 2.0 and that the ratio of enzyme/inhibitor at 100% inhibition was 1.06 for pepstatin and 1.12 for the iodinated pepstatin. Equilibrium
Dialysis
The results of the equilibrium dialysis experiments obtained with the equation of Pepsin Activity Assay Johnson and Knowles (7) are summarized in It was established that the reaction of Table I. The KD obtained with pepstatin pepsin with hemoglobin under the condi- was 3.49-fold greater than that with the iotions specified was linear with time; and the dinated derivative (P < 0.004), suggesting absorbance of the supernatants after a lo- that the latter combined somewhat more
162
WORKMAN AND BURKITT
INHIBITOR
CONCENTRATION
X lOa MIL
FIG. 5. Inhibition of pepsin by pepstatin and pepstatin-L-iodotyrosine methyl ester. The residual proteolytic activity of a 1.22 x 10m7M solution of pepsin was assayed with bovine hemoglobin as substrate. For each inhibitor the correlation coefficient, slope, and intercept of the data were calculated by the method of least squares: pepstatin (O), r = -0.99418, m = -0.10073, x: intercept = 1.15 X 10-’ M; pepstatinL-iodotyrosine methyl ester (A), r = -0.99260, m = -0.10016, x intercept = 1.095 x 10m7M.
strongly with pepsin at pH 5.0 than did pepstatin itself. The data were also analyzed by plotting lliouT vs e/i,, (Fig. 6), a linear regression which according to Nimmo et al. (16) tends to minimize random errors. This analysis tended to emphasize the difference between the dissociation constants which had been determined with the equation of Johnson and Knowles (‘7). From these results it definitely appeared that pepstatinL-iodotyrosine methyl ester was at least as good an inhibitor of pepsin as is pepstatin itself and may even have bound more tightly by a factor of 3-14.
FIG. 6. Determination of the K,, for pepsin-pepstatin and pepsin-pepstatin-L-iodotyrosine methyl ester by equilibrium dialysis. The results of equilibrium dialysis are plotted as l/i,,r,r against e/i,, for pepsin-pepstatin (0) and pepsin-pepstatin-L-iodotyrosine methyl ester (A). The data were analyzed by the method of least squares. The K,, for each complex is equivalent to the inverse of the slope ?rtor 1.38 x 10. ‘” M for the former and 9.83 x 1Om’2M for the latter enzyme-inhibitor complex.
DISCUSSION
The past decade has witnessed the important development of specific inhibitors of the acid proteases. Of all the inhibitors described pepstatin is the most potent by several orders of magnitude, the K, or Ki having been reported to lie within the range of lo-‘to lo-” M for pepsin (9- 12,21), renin (ll), and cathepsin D (8). The unique inhibitory capacity of pepstatin can probably be
TABLE I DETERMINATION OF K, FOR PEPSIN-PEPSTATIN AND PEPSIN-PEPSTATIN-L-IODOTYROSINE METHYL ESTER BY EQUILIBRIUM DIALYSIS
Pepstatin-L-iodotyrosine methyl ester (1.04 X It9 M)
Pepstatin (1.02 X lo-' M)
Inhibitor species Pepsin concentration x 1OmyM
2.5
1.2
0.62
0.31
2.5
1.2
0.62
0.31
K,
6.68
4.10
3.62
3.86
0.93
1.19
2.23
0.89
x
lo-”
M'"
Mean f SD
4.57 5 1.42
n Calculated using the equation of Johnson and Knowles K, = i,,&
1.31 k 0.63 - Ai)/Ai.
PEPSIN
INHIBITION
BY IODINATED
largely attributed to the two statine (4amino, 3-hydroxyl, 6-methyl heptanoic acid) residues within the molecule (12, 18), and pepstatin seems to inhibit the acid proteases at a ratio of enzyme/inhibitor ranging from 1.0-2.0 depending upon the pH (10, 12). Due to the tremendous inhibitory potential of pepstatin many applications for this compound can be envisioned in the study of the acid proteases. Consequently, it would be very useful to have a radiolabeled pepstatin which would be obtainable at an accurately known specific activity. The major underlying assumption would be that in radiolabeling the pepstatin molecule, the inhibitory characteristics of native pepstatin towards the acid proteases would be unchanged. In contrast to the relatively low specific activity pepstatin derivatives which have been prepared in the past (8-10, 14), this report describes the synthesis of an iodinated pepstatin of very high specific activity. Due to the fact that iodine-containing compounds are retarded on Sephadex gels, this property has been exploited to obtain iodinated pepstatins (with both stable and radioactive isotopes of iodine) of very high purity and/or specific activity. The identity and purity of the radioiodinated pepstatin was confirmed by its coelution from LH20 in aqueous methanol and from its identical migration in three tic systems with the nonradioactive pepstatin-L-iodotyrosine methyl ester. Quantification of the radioactivity seen in radioautographs of the tic plates revealed that virtually all of it was localized to the pepstatin-L-[1251]iodotyrosine methyl ester. The important requirement that the inhibitory properties of radiolabeled pepstatin toward the acid proteases remain unchanged has been demonstrated with the iodinated derivatives. Peptic activity was assayed with hemoglobin as a substrate to compare the behavior of pepstatin and pepstatin-L-iodotyrosine methyl ester with each other. Previous modifications of pepstatin, including esterification with methyl (8, 21), ethyl (21), and p-bromophenacyl groups (21), conversion to the amide (21), and coupling to diaminoethane (8) and glytine (8), altered the activity of pepstatin toward pepsin and cathepsin D either not
PEPSTATIN
163
at all or only slightly. In agreement with these findings, both pepstatin and pepstatin-L-iodotyrosine methyl ester generated almost identical regressions of v (or absorbance) on inhibitor concentration (Fig. 5). In addition, the stoichiometry of the reaction could also be estimated by extrapolating the inhibition curves to 100% inhibition or 0% enzyme activity at pH 2.0. The ratios of enzyme/inhibitor were 1.06 for pepstatin and 1.12 for pepstatin+iodotyrosine methyl ester. Kunimoto et al. (lo), using the synthetic peptide substrates N-acetyl-L-phenylalanyl+diiodotyrosine at pH 2.0 and Phe-Gly-His-Phe(NO,)-PheAla-Phe-O-Me at ph 4.0, concluded that the reaction of pepstatin with pepsin was equimolar, and used this information to titrate pepsin activity against known amounts of pepstatin to calculate enzyme concentration. Marciniszyn et aZ. (12) came to a somewhat different conclusion since they demonstrated a variation in the stoichiometry of the reaction with pH. These authors postulated that the stoichiometry may vary from 1 to 2, especially at pH 2.0 where each statine residue may combine with an enzyme molecule. Using intact pepstatin and hemoglobin as substrate, the ratio of enzyme to pepstatin was 1.96 at pH 2.1, 1.33 at pH 3.1, and 1.13 at pH 3.65. Using a pepstatin fragment N-acetyl-valyl-statylalanyl-statine and N-acetyl-L-phenylalanylL-diiodotyrosine as substrate, the enzyme/ inhibitor ratio varied at random from 1.61 to 1.89 over a pH range of 2.1 to 4.5. In the experiments reported here peptic activity was assayed at pH 2.0. Our results tend to support Kunimoto’s conclusions that the pepsin-pepstatin reaction is equimolar and that any deviation from this ratio is due to inactive enzyme. Our findings also demonstrate that the inhibitory capacity of pepstatin was essentially unchanged by the chemical modification at its carboxyl terminus. An accurate determination of either the Ki by kinetic methods or the K, by equilibrium dialysis would provide the most precise estimate of the strength of the pepsinpepstatin interaction. The interpretation of kinetic studies is limited, however, by the fact that the traditional substrates for pep-
164
WORKMAN
AND BURKITT
sin technically cannot be used within the concentration range of the Ki. The availability of high specific activity radioiodinated pepstatin which could be diluted to trace quantities at precisely calculated specific activities with nonlabeled pepstatin species permitted the performance of equilibrium dialysis at a much lower concentration range of pepsin and pepstatin. Both the equation of Johnson and Knowles (7) and the linear regression of l/ZoUT vs e/i,, of Nimmo et al. (16) were used to calculate the K,. The values obtained indicated that under the conditions specified, pepstatin-Liodotyrosine methyl ester actually appeared to form a more stable complex with pepsin than did native pepstatin. These values agreed very well with the Ki of 9.7 x lo-” M for the pepsin-pepstatin complex obtained by Kunimoto et al. (10) using the synthetic substrate Phe-Gly-His-Phe(NO,)-Phe-Ala-Phe-O-Me at pH 4.0, but were considerably lower than the values of 1.2-1.7 x 1O-gM determined by McKown et al. (11) using an ‘251-labeled synthetic polymeric substrate at pH 5.5 and 1.1 x 10egM found by Marciniszyn et al. (12) using hemoglobin as substrate at pH 2.1 and assuming that two pepsin molecules combine with each pepstatin molecule at this pH. The equilibrium dialysis studies provide further evidence that the radiolabeled pepstatin behaves towards pepsin essentially in the same manner as does native, unmodified pepstatin. This finding suggests that radioiodinated pepstatin might be useful as an affinity label in the study of the entire class of acid proteases. ACKNOWLEDGMENTS We are greatly indebted to Dr. Tadashi Inagami for his many helpful suggestions and review of the manuscript. REFERENCES 1. AOYAGI, T., MORISHIMA, H., NISHIZAWA, R., KUNIMOTO, S., TAKEUCHI, T., UMEZAWA, H., AND IZEKAWA, H. (1972) J. Antibiot. 25. 689-694.
2. BARRETT, A. J., AND DINGLE, J. T. (1972) Biothem. J. 127, 439-441. 3. FERGUSON, J. B., ANDREWS, J. R., VOYNICK, I. M., AND FRUTON, J. S. (1973)J. Biol. Chem. 248, 6701-6708. 4. GROSS, F., LAZAR, J., AND ORTH, H. (1972) Sciewe 175, 656. 5. GUYENE, T. T., DEVAUX, C., MENARD, J., AND CORVOL, P. (1976) J. Cl&. Endocrino~. 43, 1301-1306. 6. HUNTER, W. M., AND GREENWOOD, F. C. (1962) Nature (London) 194, 495-496. 7. JOHNSON, C. H., AND KNOWLES, J. R. (1966) Bzothem. J. 101, 56-62. 8. KNIGHT, C. G., AND BARRETT, A. J. (1976) Bio&em. J. 155, 117-125. 9. KUNIMOTO, S., AOYAGI, T., MORISHIMA, H., TAKEUCHI, T., ANDUMEZAWA, H. (1972)J. Antibiot. 25, 251-255. 10. KUNIMOTO, S., AOYAGI, T., NISHIZAWA, R., KOMAI, T., TAKEUCHI, T., AND UMEZAWA, H. (1974) J. Antibiot. 27, 413-418. 11. MCKOWN, M. M., WORKMAN, R. J., ANDGREGERMAN, R. I. (1974) J. Biol. Chem. 249, 77707774. 12. MARCINISZYN, J., HARTSUCK, J. A., AND TANG, J. (1976) J. Biol. Chem. 251, 7088-7094. 13. MILLER, R. P., POPER, C. J., WILSON, C. W., AND DEVITO, E. (1972) Biochem. Pharmacol. 21, 2941-2944. 14. MORISHIMA, H., SAWA, T., TAKITA, T., AOYAGI, T., TAKEUCHI, T., AND UMEZAWA, H. (1974) J. Antibiot. 27, 267-273. 15. MORRISON, J. F. (1969) Biochim. Biophys. Acta. 185, 269-286. 16. NIMMO, I. A., ATKINS, G. L., STRANGE, R. C., AND PERCY-R• BB, I. W. (1977) Biochem. .J. 165, 107- 110. 17. ORTH, H., HACKENTHAL, E., LAZAR, J., MIKSCHE, U., AND GROSS, F. (1974) Circ. Res. 35,52-55. 18. RICH, D. H., SUN, E., AND SINGH, J. (1977) Biothem. Biophys. Res. Commun. 74, 762-767. 19. RYLE, A. P. (1970) in Methods of Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 316-336, Academic Press, New York. 20. SUBRAMANIAN, E., SWAN, I. D. A., Lru, M., DAVIES, D. R., JENKINS, J. A., TICKLE, I. J., AND BLUNDELL, T. L. (1977) Proc. Nat. Acad. Sci. UISA 74, 556-559. 21. UMEZAWA, H., AOYAGI, T., MORISHIMA, H., MATSUZAKI, M., HAMADA, M., ANDTAKEUCHI, T. (1970) J. Antibiot. 23, 259-262. 22. WOESSNER, J. F. (1972) Biochem. Biophys. Res. Commun. 47, 965-970.