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Purification and Characterization of a Trypsin Inhibitor from Solanum tuberosuml DPpur,trme~ltde Biochimie, Fucr~lrPde Mlrlecilre, UlliversitP rle Sherbrooke, Skerbrooke, QuPbec J 1 H 5N4 Received July 2, 1974 Rouleau, M. & Lamy, F. (1975) Purification and Characterization of a Trypsin Inhibitor from Solunu~?~ tuberosum. Cull. J. Biochem. 53, 958-974 A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole, T o a lesser extent it combines also with chymotrypsin and elastase. For trypsin, Ki = 8 X M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains 110 sugar or free sulfhydryl groups. Its extinction coefficient EZBo1% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22 000, as determined by sedimentation equilibrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichiometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physicochemical methods. Rouleau, M. & Lamy, F. (1975) Purification and Characterization of a Trypsin Inhibitor from Solu~~um tuberosum. Cun. J. Biochern. 53, 958-974 Un inhibiteur de trypsine a kt6 isole a partir d'une poudre acetonique de pomme de terre (Sola~lumtuberosum) et purifie par chromatographie d'affinite. Cette proteine, en plus d'inhiber la trypsine dans un rapport 1:1, inhibe egalement, mais a un degre moindre, la chymotrypsine et l'elastase. La valeur de la constante d'inhibition de la trypsine (Ki) est etablie a 8 X M. Cet inhibiteur semble constitue d'une seule chaine polypeptidique de 207 acides aminks. 11 ne contient aucun sucre, aucun groupement SH libre. Son coefficient E2s01% est 10.3 et son point isoelectrique (pl), 6.9. Son poids moleculaire, evalue par ultracentrifugation, composition en acides aminks et pouvoir inhibiteur de la trypsine est de l'ordre de 21 000 a 22 000. L'homogeneite de cette proteine est confirmee par electrophor&se a deux pH, par ultracentrifugation et par les experiences d'inhibition. L'identification de deux acides amines en position N-terminale, jointe a la stoichiometrie variable de l'inhibition des differents enzymes proteolytiques soukve la possibilite d'une contamination par un inhibiteur polyvalent non detectable par les techniques physicochimiques utilisees.

Introduction The presence of proteinase inhibitors in plant material is well documented (1, 2) ; their physiological role, which is not clear, could be important. Ryan (3) has suggested that they could be regulatory agents in the control of endogenous proteinases, or protective agents directed against insect and /or microbial proteinases. They could also represent storage proteins. In potatoes (Solanrrnz tu berosuin), inhibitors of trypsin (4-6), chymotrypsin (7), elastase (8, 9), kallikrein ( lo), carboxypeptidases A (1 1) 'This work was supported by the Medical Research Council of Canada (grant MT-4003). "resent address: Laboratory of Pathophysiology, National Institutes of Health, Bethesda, Maryland 2001 4. 3 T whom ~ inquiries should be addressed.

and B ( 12), and of different proteases (2) have been identified and some of their properties reported. In the case of potato trypsin inhibitors, a constant finding has been that they seemed to exist in a multiplicity of molecular forms (4-6) differing in their chemical and biological properties. For example, their amino acid composition varied. In particular, the half cystine content was reported to be between 1 and 12y0 (5, 6, 13). The N-terminal residue was found to be Ala (13), Glu, Ala, and Asp (6) or Arg (5). The molecular weight, on the contrary, was usually reported as being about 20 000, although Iwasaki et al. (13) interpreted this value as that of a dimer. From a biological standpoint, the specificity of these inhibitors was variable. Among the thirteen

ROULEAU AND LAMY: CHARACTEWHZATION OF A TRYPSIN INHIBITOR

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molecular species detected by Belitz et al. (51, two inhibited only trypsin, while the remainder could combine with trypsin and chyrnotrypsin. Similarly, Iwasaki et al. (14, 15) have characterized two inhibitors having the same molecular weight, the same amino acid composition and N-terminal groups, and the same reactive site. Yet one of them inhibited chymotrypsin and a commercial preparation of subtilisin BBN' (Nagarse) while the other was polyvalent toward trypsin, chymotrypsin, and Nagarse. TO understand this complexity it seems necessary to rigorously purify different inhibitors and to carry out detailed study of their structure. As a first step toward this goal, we report in this paper on the purification of potato trypsin (EC 3.4.21.4) inhibitor by affinity chromatography. It appears to be pure by many criteria. Wowever, a more detailed study suggests that this might not be the case. The purified preparation is suspected to contain at least two proteins, both inhibiting trypsin mole per mole but one of them capable also of combining with chymotrypsin.

Potatoes (Solanunt t~~bcrssuraa) were purchased from a local supermarket. Their variety was not determined.Three different batches, grown in Maine, New Brunswick, and Prince Edward Island, were used. Cyanogen bromide, h n s y 1 chloride, and constantboiling hydrochloric acid 6 A4 (Sequanal grade) were from Pierce Chemicals. Ampholine was obtained from LKBProdukter AB, Sweden; from Cyclo Chemical; and HNTSAS, from Eastman Chemical Co.; ATEE and BAEE were from Sigma Chemical Co.; Polyamide sheets were purchased from Gallard-Schlesinger Chemical Co. ; Polyclar-AT (an insoluble polyvinyl pyrrolidone) was a gift from Chemical Developments of Canada; Casein 'Hammersten' and Trypsin 1-3CM) were from Nutritional Biochemicals Corporation. Elastin was purified from ligamentum nuchae according to Lansing e6 ul. (19). 4The following abbreviations are used: HNTSAS, OW-2-NO2-5-toluenesulfonic acid sultone; NPGB, gnitrophenyl-p-guanidino-benzoatehydrochloride; ATEE, N-acetyltyrosineethyl ester ; BAEE. benzoylarginineethyl ester; NATAME, N-acetyltrialanylatemethylester; DTE, dithioerythritol; PAGE, polyacrylarnide gel electrophoresis; TU, trypsin units by the potentiornetric method (16); TU(SF), trypsin units by the spectrophotometric method sf Schwert-Takanaka (17); CU, chymotrypsin unit by the potentismetric method (18); EU, Elastase unit; n = x , after a numerical value, followed by a standard deviation, indicates the number of experimental determinations.

Methods rCleasuratrte~~ts of Protein Cotzcentration We measured the extinction cmficient ( E 2 s ~ ' %of) the inhibitor. Amounts of salt-free lyophilized inhibitor varying between 0 and 1 mg were weighted on a Cahn electrobalance and dissolved in 1.0 ml of 0.05 rVf phosphate buffer, pH 7.0. From the absorbancies of the various solutions we calculated E 2 8 0 1 % = 110.3 0,1 ( n = 10). The concentration of elastase (EC 3.4.21.15) was calculated using an E2801%of 20.2 (20). The concentrations of trypsin and chymotrypsin were expressed in terms of active site concentrations. Trypsin was titrated with NPGB following the method of Chase and Shaw (21). A molecular weight of 23 306 (22) and an Ezscl%of 15.4 (16) were used for the calculations. Six different determinations indicated that 55.2 F 0.8(,'/, of the trypsin (on a dry weight basis) was active. Chymotrypsin was titrated with HWTSAS following the method of Kezdy and Kaiser (23). For the caIculations we used a molecular weight of 25 590 (24) and an E2s01% of 20.5 (16). By saturating the reagent with the enzyme, we determined a value for the extinction coefficient of the sulfonyl enzyme ( E ~ ~ of, ) 7375 76 (it = 4). By saturating the enzyme with the reagent, we established that the chymotrypsin was 91.2 2 2.576 active (to = 9).

+

+

rVIetasureiineltts cf Enzymatic Activity Two assays were used for the determination of the trypsin activity. The first one was a pH-stat potentiometric assay using BAEE as substrate, as described by Walsh and Wilcox (16). A trypsin unit (TU) is that amount of trypsin which hydrolyzes 1 pmol of BAEE per minute at pH 8.0 and 37 "C. Under these conditions, trypsin had an activity of 128 9 1 TU (18 = 12) per milligram of active enzyme. The second method routinely used was a spectrophotometric one developed by Schwert and 'Fakenaka (17) and modified by Greene et aH. (25). The variation of the absorbancy of the substrate (BAEE) was monitored at 253 nm using a recording sgectrophotometer. One trypsin unit (TU(ST)) is defined as that amount of trypsin which causes a change of 8.8 absorbancy unit per minute at pH 8.0 and 20 "C. Under these conditions, trypsin had an activity of 61.5 9 0.5 TU(ST) per milligram of active enzyme (I! 15). The routine determinatioil of the activity of chyinotrypsin was performed pstentiometrically on a pH-stat with ATEE as substrate as. described by Uram and Lamy (18). One chynlstryptic unit (CU) is defined as that amouilt of the enzyme which hydrolyzes 1 prmolof ATEE per milligram at pH $.Q and 37 "C. Under these casnditions, chymotrypsin had an activity of 802 12 CU per milligram of active enzyme 01 12). The elastinolytic activity of elastase was measured according to the method of Ardelt et al. (26). To 3.0 ml of 0.05 M Tris buffer, pH 8.6, we added 50 mg of elastin and varying amounts of elastase (14-140 pg). After an incubation time of 20 min at 34 "C, the reaction was stopped by the addition of 5 ml of 0.1 N NaOH. The non-hydrolyzed elastin was centrifuged and the absorbancy of the supernatant measured at 276 nm. An elastinolytic unit (EU) is defined as that amount of elastase which hydrolyzes 1 mg of elastin in 20 min at pH 8.6 and 37 "C. Under these conditions, elastase had an

-

-

+

960

CAN. J. BIOCHEM, VOk. 53, 1975

activity of 40.0 + 0.3 EU per milligram sf enzynle (m 18). In some experiments, the non-specific proteolytic activity of potato extracts was measured by the casein method of Kunitz (27). The possible elastase activity of these extracts was tested with NATAME as substrate, following Gertler and Hofmann (28).

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LAeasuremerntsof the Odribitory Activity For the assay of the inhibitory activity, the inhibitor and the enzyme were dissolved in the appropriate buffer for the test. To a constant amount of active enzyme we added different amounts of inhibitor. After 10 rnin preincubation, the residual enzymatic activity was determined as described above and the concentration of free active enzyme calculated. The inhibitory activity was taken to be the difference between the total and the residual enzymatic activity. While the hydrolysis of BAEE by trypsin alone was linear with time, the presence of inhibitor in the assays n~odifiedthe kinetics. With time the rate of hydrolysis of BAEE increased as if more free enzyme became available. In this case the initial rate of the reaction was measured by graphical extrapolation t o zero time of the curve relating either ~ A ~ ~ ~ or l m thei namount of NaQH consumed per minute to time. In the case of chymotrypsin, the hydrolysis of ATEE proceeded linearly with time in the absence as well as in the presence of inhibitor. Since elastase activity was measured by the amount of peptide released from elastin at a single time, we have no information on the kinetics of the enzyme. A~~alyticnl Metho& For amino acid analysis, protein samples containing approximately 0.1 rng and a known amount of norleucine were hydrolyzed at 105 "C with constant-boiling hydrochloric acid in evacuated sealed tubes for 24, 48, 72, and 96 la, respectively. Analyses were performed using a Technicon amino acid analyzer, model TSM-1. The buflers used were those recommended by Ertingshausen et ul. (28) followed by a 8.07 N borate buffer, pH 9.4, containing 0.3 M NaCl and ]IC>; BBJRHJ-35. Each analysis lasted 80 rnin. All values for the amino acids were corrected for the yield of norleucine. Tryptophan and tyrosine were determined by the method of Goodwin and Morton (30) and by that of Edelhoch (31). The p-chlorornercuribenaoate method of Boyer ((32) was employed for the determination of free sulfhydryl groups; analyses were performed in 0.33 M acetate buffer, pH 4.6. Disulfide bonds were titrated by the method of Iyer and Klee (33) in which the oxidation of dithioerythritol was followed at 310 ram. The cysteine - sulfuric acid and a-naphtol reactions (34) were used for carbohydrate detection. N-terminal residues were detected after dansylation of the protein, hydrolysis, and two-dimensional chromatography on polyamide sheets (5 X 5 sm), according t o the method of Woods and Wang (35). This determination was verified by several preliminary experiments kindly performed by Dr. David Gibson ow an IlBitrsn sequenator. The method, together with the techniques of alkylation and reduction of disulfide bonds. are described in detail by Gibson (36).

Electrophoretic Methods Hsoelectric focusing experiments were performed on an LKB apparatus (column LKB-8800-10) according to the instructions of Haglund (37). The gradients, stabilized by a continuous sucrose gradient (0-507;), varied either between pH 3 and 18 or between pH 6 and 8. The runs were carried out at 4 O C , at 700 V and lasted 60-70 h. Fractions of 8.4 ml were collected. The protein was detected at 280 nm and the pH of every fifth tube was measured at room temperature. Polyacrylamide gel electrophoresis was performed using the solutions described by Maurer (38). Basic runs at pH 8.9 were made in a Tris-glycine buffer, while the acidic ones used p-alanine - acetic acid. The electrophoresis took place in 15'j;, gel columns, 6.0 can high, with a diameter of 0.6 cm; 3 mA per tube were applied for 30 min. The current was then raised to $ mA per tube for 1.5 h. At the end of I e runs, the gels were fixed and stained in Coomassie Blu (39). Ca!c~~batiotzs of bke Dissscisrtiorr Cortsta~ttjar. Trypsin-Hmzhibitor Complex The approxinrate dissociation constant (Ki) for the I E H (E, free enzyme; 1, free inequilibrium EH ; hibitor) was calculated according to the method of Green and Work (40). A mere accurate value of Ki was also established by a curve-fitting technique using a PDP-9 digital data computer. From the equation describing a simple equilibrium, we can express Ki in functioi~sf E (concentration of free enzyme), E, (total enzyme concentration) and 1, (total inhibitor concentration) as follows :

+

Knowing E, (a constant) and assuming diKerent values for Ki,we can calculate E corresponding to the experimental concentrat ions of I,. The computer program then compared the calculated values of E (E,) with the expaimental ones (Ee) shown in Fig. 9. The best value of Ktwas that for which the error function

was minimum. Afiniq). Chroma tograp/gl

Bovine trypsin was covalently fixed to CNBr-activated Sepharose-4B. following the method of Kassell and Marciniszyn (41). The coupled resin was poured into a column and washed with I litre of 8.1 M carbonate buffer, pH 9.0, then with 1 litre of 1.0 M NaCl and finally with a 0.05 hl acetate buffer, pH 4.5. The total Az8, of the effluents indicated a yield for the coupling reaction of 52, 53, and 92$$ for three different experiments. Deactivated Sepharose was prepared by lettin, $e CNBr-activated Sepharose in a cold room at pH 9.6 ~ u r 24 h. This resin was then equilibrated with 0.05 hf acetate buffer, pH 4.5, and used several weeks later. Ul frucerztrifugation &18tracentrifugationexperiments were performed with a Beckman model E ultracentrifuge equipped with interfer-

ROULEAU AND LAMY: CHARACTERIZATION OF A TKYPSZN INHIBITOR

96 1

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TABLE1. Inhibition of trypsin, chyn~otrypsin,and ence optics and an electronic automatic speed control. High speed equilibrium data (42) were measured from the elastafie by acidic and alkaline crude extracts usual Rayleigh interference photographs and the average molecular weight was calculated from a graph relating In Inhibition bya C to r2,where Cis the concentration at a radial distance r. Acidic extract Alkaline extract When there was indication of non ideality in the solution, we computed the molecular weight from the Trypsin 0.46b experimental data by Method 111 of Rowe and Rowe Chymotrypsin 8.20 (43). This method is based on the equation Aln C/AC = Elastase 0.16 (ko/2) Ar2/AC a where, in addition to the usual aExpressed as milligrams of enzyme inhibited per milligram of symbols, ko = M o ((I - F P ) / R T ) ~a? , = I: Sln -y/6C and extract. -/ is the activity coefficient of the different components of bFigure 9 shows the type of curve obtained in inhibition exprrithe system. The runs lasted 24 h at 40 000 rpm at 20 "C. anents. Only the linear portion of the curves is used for the ca~culations. The protein, at a concentration of approximately 2.0 rng/ml was thoroughly dialyzed against the chosen buffer the precipitate was collected by centrifugation, before the experiment. The partial specific volume (7) of dissolved in 500 rnl of H 2 0 , and dialyzed exthe protein in the buffer was taken to be 0.7212 ml/g, a tensively against H20. The solution was then value obtained from amino acid c' nposition by assuming single additivity of the specific v umes of the amino acid centrifuged and lyophilized. Six to seven grams residues (44). In presence of 6 M guanidinium-HC1 an of white powder were obtained. We will refer empirical value of (1 - Fp) of 0.162 (45) was used to to this fraction as 'the acidic crude extract'. calculate the molecular weights. The insoluble material collected on the cheese

-+

cloth during the acidic extraction was further treated with 3 litres of 0.25 A4 borate buffer, Preparation of Crlrde Extract o f Proteins from pH 8-Oy containing 170NaC1- After filtration on cheese cloth and centrifugation, the supernatant Potatoes and was treated as described above for the acidic ~~~~h potatoes were homogenized with D~~rce - cooled acetone a extraction. Three to four grams of white powder Waring blendor. ~h~ homogenate was filter& on were obtained and thereafter called 'the alkaline Whatman filter paper No. 4. The color of the crude extract'. filtrate varied from yellow to green. Hornog- Properties of the Acidic and Alkaline Cridde Extracts enization and filtration were repeated once. The granulous paste thus obtained was washed on 3'0 remove the phenolic compounds, both the the filter paper with cold acetone until the filtrate acidic and the alkaline extracts (20 mg in 2 ml became colorless. The paste was then dried at 0.1 A4 acetic acid) were chromatographed on a room temperature. Approximately 640 g of a column (75 X 2.5 cm) of PolycIar-AT, prelight tan acetone powder were obtained from 18 viously equilibrated with the same solvent. The lb of potatoes. In some extractions, blackening protein, eluted with 0.1 M acetic acid, emerged of the powder occurred during drying, indicating in a single peak corresponding to 100% of the a poor extraction of the phenolic compounds. initial A280.This indicated that the extracts conSuch a powder was discarded as a starting tained no phenolic compounds (46). material. The extracts were compared by polyacrylaAll subsequent operations were carried out at mide gel electrophoresis at pH 4.5 (see the two 4 "C. The centrifugations were always per- gels on left of Fig. 2). Six bands were found formed in an International centrifuge model in each extract. The mobilities of the correspondB-20 (rotor No. 877) at 12 880 rpm for 15 min. ing bands were indistinguishable. However, the The acetone powder (640 g) was extracted relative concentration of each protein differed for 24 h with 3 litres of 3y0 (v/v) acetic acid in each case. con$-;-ling 174, NaCZ. The final pH was between Both extracts had no proteolytic activity 3 4. After removal of the insoluble material against casein at pH 7.6, 8.0, and $.6 or against by filtration through four layers of cheese cloth, elastin at pH 8.6. No esterase activity could the opalescent filtrate was clarified by centrifuga- be detected at pH 8.0 using ATEE4, BAEE, and tion. To the limpid pale-yellow supernatant, NATAME as substrates. solid ammonium sulfate was added to give $00/, Both extracts contained inhibitors of trypsin, saturation (0.536 g/ml. After standing overnight chyrnotrypsin, and elastase. Table 1 summarizes

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CAN. J. BIOGHEM. VOL. 53, 1975

FIG.I. Affinity chromatography of an acidic extract sf a potato acetonic powder (600 rng in 25.0 rnl of acetate buffer, 0.05 M , pH 4.5). Panel A, elution with 0.05 M acetate bufkr, p H 4.5; panel B, with 1.0 M NaCl; panel C, with 0.012 M HC1- 0.01 CaClp.Aliqusts of 19.4 ml were colIected at a flow rate of 6Q rnl/R. The horizontal bar under each peak indicates the factions which were poded, dialyzed, and lysphilized.

column (2.5 X 17.5 cm) prepared as described in 'Methods'. Seventy-nine percent of the initial was eluted as a, large composite peak by the acetate buffer. A solution sf $.OM urea, pH 2.0, or 1.0 M NaCl eluted a second peak of protein containing 2%yo of the original protein. PoByacrylamide gel electrophoresis of this fraction showed six bands identical with those present in the crude extract. The solution of A finitgr Chrorzzatography of eBze Crude Extracts 0.012 M HCl - 0.01 M Ca@12used t s elute the inhibitor from the Sepharose-trypsin column on a Seph~rose-TrypsinCslharnn Two control experiments were performed to did not release any protein from that column. Both the acidic and the alkaline crude extracts check that the presence of covalently attached enzyme was responsible for the retention of pro- were chromatographed on a Sepharose-trypsin teins on the column of Sepharose-trypsin. First column (2.5 X 42 cm). The results were similar the crude acidic extract (5 m1 in 0.05 M acetate and we shall describe a purification from an buffer, pH 4.5; 9.65 A2,,/rnl) was filtered acidic extract. Six hundred rniIligrams of acidic through an unmodified Sepharose column (1.8 extract were dissolved in 25 ml of 0.05 A4 aceX 48 cm). Elution was carried out with the tate buffer, pH 4.5, and chrornatographd. The same bulffer, and a single symmetrical peak column was washed with the acetate buffer until emerged containing 10B~eof the initial optical the absorbance at 288 nm was 0.850 and then density units. For the second control experiment, with unbuffered 1.8 M NaCI. When the A28Q we chromatographed the crude acidic extract reached a value sf 0.02, the inhibitor was eltutd (5 rnl in 8.05 M acetate buffer; 18.14 A280/ml) with 8.812 M HCl - 0.01 M CaC12. The elution on a deactivated CNBr-activated Sepharose pattern is shown in Fig. 1. The five peaks eluted

the results. The inhibitory activity sf both extracts was, in general, very similar. However, the acidic extract was twice as active against trypsin as the alkaline extract, the latter being prepared from the residue sf the acidic extract. Since no real difference between the extracts could be detected, we used either one for the further purification of the trypsin inhibitor,

963

RBULEAU AND LAMY: CHARACTERIZATION OF A TRYPSIN INHIBITOR

TABLE 2. Inhibition of trypsin and chymstrypsin by the different peaks obtained by affinity chromatography on a Sepharose-trypsin column@

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Inhibition s f chymotrypsinb

Peak No.

Proteins assayed,

Enzyrne inhibited,

Pg

%

Inhibition of trypsinc

Specified inhibitory activity

Proteins assayed,

Enzyme inhibited,

M

Pg

Specificd inhibitory activity

OThese assays were performed as described in 'Methods' but at only one concentration of inhibitor. Therefore, the values given are to be considered as approximate. bThe amount of chymotrypsin used in these assays was 13-1 rg of active chymotrypsin. T h e amount o f trypsin used in these assays was 9.4 pg of active trypsin. dMil1igrarn.sof enzyme inhibited per milligram of protein assayed.

FIG.2. Polyacrylamide gel electrophoresis at pH 4.5 of: A, alkaline extract; B, acidic extract; C-G, pooled fractions 3-7 as defined in Fig. 1.

by acetate (peaks 1-5) and the single peak (peak 6) eluted by Na61 contained, respectively, 67.3 t 3.7% and 15.4 t 1.57, of the total pro' tein ( n = 8). The inhibitor (peak 7) represented 17.1 r_t 2.6y0 of the initial protein (n == 8). It is to be noted that this percentage was constant whatever the initial load of the column (varying betweer, 300 and 800 mg). This indicated that the column operated well within its maximum capacity. The fractions containing each peak were pooled according to the scheme of Fig. 1 and concentrated on a Diaflo Amicon ultrafiltration system. No proteins passed through the UM-10 membrane. The concentrates were

dialyzed extensivelyagainst water and lyophilized. Table 2 shows the inhibitory activity of the different peaks against chymstrypsin and trypsin. Peaks 1-3 have essentially no activity. When compared with the activities of the acidic extract (Table 1), the proteins contained in peaks 4-6 showed an increased activity against chymotrypsin but essentially the same activity against trypsin. The inhibitor peak (peak 7) was very active against trypsin while its activity against chyn~otrypsinhad not changed (see Table 1). Poliyacrylamide gel electrophoresis were performed on each peak and the electropherogran~s are shown in Fig. 2. Peaks 1 and 2 gave no

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FIG. 3. Polyacrylamide gel electrophoresis at pH 4.5 and 8.9 of different preparations of inhibitor purified by affinity chromatography (peak 7 of Fig. I). Inhibitor purified: A, from an acidic extract of an acetone powder from potatoes grown in New Brunswick in 1973 ; B, from an alkaline extract prepared from potatoes grown in Maine in 1972.

band. Peaks 3-5 were obviously polydisperse, since at Beast two bands could be seen. Only peaks 6 and 7 seemed pure. Peak 7 which had the maximum antitryptic activity was more cationic than peak 6 which h antichyrnotryptic activity. The mobilities of the bands were measured. The protein in peak 7 proved to be distinct from those present in the other peaks. Sugars were qualitatively determined by the a-naphtol test (34). Only peaks 1 and 2 showed a strong positive reaction. All the other peaks were entirely negative. EHectsopBaoresis of the Inhibitor Purified by Afinn'ry Chro~notogroph-y Figure 3 shows the electropherogrms of two purified inhibitor preparations, one obtained from an acidic crude extract and the other from an alkaline one. The electrophoreses were carried at pH 4.5 and 8.9. No obvious evidence of polydispersity was displayed and proteins with similar electrophoretic properties were purified. This was true whatever the geographical origin or the year of the crop of the potatoes from which the inhibitor was isolated. Figure 4A presents the results of a pH gradient electrophoresis (pH 3-10) of the purified inhibitor prepared from an acidic extract. A main

peak having an isoelectric point of 6.9 was present. Several other peaks were evident especially at pH 8.4, 6.4, and 5.5. All these peaks showed inhibitory activity against trypsin. Fractions 193 1, containing proteins focused between pH 7.6 and 6.4, were pooled and rerun on a pH gradient 6.0-8.0. The pattern obtained is shown on Fig. 4B. It is clear that most of the secondary peaks were eliminated and that a single protein (pb = 6.9) was present in high concentration. In every purification we obtained a major component with a pB of 6.9, In some cases it repres approximately 50&r,of the protein pre purified inhibitor (Fig. 4A). More often it amounted to almost 90% (Fig. 4C) and such re chosen to carry out the experiMolecular Weight Deteminations by UHtracmtrif ugation When the experiments were performed in 0.1 M NaCB or in 6.0 M guanidinium chloride, both with and without mercaptoethanol (Fig. 5A), the plot of Hn C vs. a%as linear with no evidence of polydispersity. From these data a moHecuHar weight of about 21 008 could be calculated (Table 3). However, when the inhibitor was run in 0.1 M acetic acid, a curvature was observed

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ROULEAU AND I-AMY: CHARACTERIZATION OF A TRYPSIN INHIBITOR

fractions FIG.4. Isoelectric separation of the inhibitor purified by affinity chromatography. The electrophoreses were in a pH gradient from 3 to 10 on two different inhibitor preparations purified from potatoes grown in Maine in 1972 (panel A) and New Brunswick in 1973 (panel C). Fractions 19-30 (horizontal bar in panel A) were rerun on a pH gradient from 6 to 8 (panel B). Each fraction had a volume of 1.42 ml. (-) Absorbance at 280 nm, (- - -) pH of each fraction. TABLE3. Summary of molecular weight determinations of potato trypsin inhibitor Methods

Molecular weight

Ultracentrifugation In 0.1 M NaCl In 0.1 M NaCl - 0.29 M mercaptoethanol In 0.1 M acet ic acid In 6.0 M guanidinium chloride In 6.0 M guanidinium chloride mercaptoethanol Trypsin inhibition Residual enzymatic activity Residual active site titration Amino acid analysis

aMolecular weight f the standard deviation of the appropriate experimental slope (In C vs. ra in the case of ultracentrifugation; residual active enzyme versus inhibitor concentration in the case of trypsin inhibition). bThe value was estimated by application s f the method of Roweand Rowe (43). CThe value was obtained by the statistical method of Delaage (47).

(Fig. 5B) opposite to that expected from polydispersity. We considered that the inhibitor dissolved in acetic acid resulted in a non-ideal solution. Rowe and Rowe (43) have presented a theory applicable to such a case. When applied

to the experimental data (see insert in Fig. 6B), a plot of ~ l C/AC n vs. Ar2/AC was linear. The molecular weight calculated from this graph was 20 675 dz 279. Tyrosine and Tryptophan Contents of the Zlahibitor The ultraviolet absorption curve of the inhibitor was measured at pH 7 in 0.05 M phosphate buffer and at pH 13 in 0.1 M sodium hydroxide. At pH 7.0, a maximum was noted at 276 nm. At pH 13, a red shift was observed, suggesting a low concentration of tryptophan with respect to that of tyrosine. The concentrations of these two amino acids were measured spectrophotornetrically following the method of Goodwin and Morton and that of Edelhoch (Table 4). On the average, tyrosine amounted to 5.2 -+- 0.3y0 (w/w) of the inhibitor m d tryptophan 1.8 t- 0.2y0 (w/w). Thus, on the basis of a molecular weight of 21 000, the inhibitor contained 7.0 rnol of tyrosine and 2.0 mol of tryptophan. Determination of Cysteine and Cystine Increasing volumes (from 0.1 to 1.0 ml) of p-chlorornercuribenzoatesolution (7.71 x M in 0.01 M phosphate buffer, pH 7.6) were

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CAN. J. BIBCHEM. VBL. 53, 1945

FIG. 5. Sedimentation equilibrium experiments on trypsiw inhibitor. The runs were carried out at 40 000 rpm for 48 h at 20 "C. Hn panel A, the inhibitor (2 mg/ml) was dissolved in 6.0 M guanidiniurn chloride. Hn panel B, it was dissolved in 0.1 M acetic acid. The data obtained in this latter case were plotted according to Rowe and Rowe (43). See insert in panel El.

TABLE 4. Speetrophotometric determination of tryptophane and tyrosine -

-

-

Method of Goodwin and Morton (30) Experiment No. Inhibitor (mg lml)" Tyrosine (mgirnl) C7

/c

Mean % Tryptsphane (mgjml) %I

Mean %,

1

2

3

4

0.735 39.56 5.38 5.46

8.540 29.98 5.55 69-12

0.740 49.43 5.33

0.545 30.34 5.57

9.27 1.72

12.20 1.65

9.26 1.70

_+

12.64 1.72 1.70

+

0.03

UInhibitor purified by affinity chromatography (peak 7 Fig. 1).

Method of Edelhoch (3 1) 1

2

0.875 0.800 41.61 38.19 4.67 4.77 4.90 k 0.21 16.87 15.05 1.$8 8.93 1.95 k 0.11

3

4

0.770 40.15 5.21

8.465 23.01 4.94

16-35 2.12

8.77 1.89

48 h

72 h

+

96 h

Average nanomolles of residues

aEach value is the mean of four determinations. It is expressed in nanornoles the standard deviation. bNanornoles of serine, thrwnine, and tyrosine a l c u b t d by linear extrapolation at zero time. CNanomolles of waline: and isoleucine obtained at 96 h of hydrolysis. dNanomoles of tryptophan measured by spectrophotometry.

Thr Ser Glx Pro G~Y Ala CY~ Val Met Ile Leu Tyr The T~P

Asx

L,ys His Arg

24 h

Hydrolysis time

TABLE5. Amino acid composition of trypsin inhibitor

Based on six cysteines

Based on all residues (Delaage (47))

Number of residues/mole of inhibitor

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Nearest integer value

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CAN. J. BXOCHEM.

VOL. 53,

8975

TABLE 6. N-terminal residues of different trypsin inhibitor preparations determined by sequenator

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Nanomoles of amino acids detected N- terminal residues

lnhibitor obtained after affinity chromatography (Fig. 1, peak 7)

Inhibitor focusing at pH 6.9 NativeQ

Reduced and alkylatedb

Asp Glu G~Y Ala Leu aThe preparation used was that shown in Pig. 4B. The ampholine was not eliminated before the experiment. bThe preparation is that contained in the mean peak shown in Fig. 4C. The ampholine was eliminated by precipitating the protein with ammonium sulfate. CThe number sfnanomoles o f the amino acid recovered was approximately 50% o f that expected.

added to 1 ml solution of inhibitor ( I mg/ml, 2.38 X 1 0 - W on the basis of a molecular weight of 211 000) in 0.33 M acetate buffer, pH 4.6, and the volume completed to 2.0 ml with the acetate buffer. No change in absorhncy at 250 nrn was detected even with 1 ml of reagent. If only one --SH group were present per mole of inhibitor, a difference in absorbancy of 8.180 would have been observed. The disulfide bridges were titrated by B T E (0.5 14 M in 6 M guanidinium-HCI). Fifteen microlitres of this reagent were added to a find volume of I ml solution of trypsin inhibitor (1.43 mg/ml, 6.81 X M) in 0.05 M TrisHC1 buffer made 6 M in guanidinium-HC1 and 1 0 - W in EDTA. The oxidation of B T E was monitored at 3 10 nm on a Cary 118-C spectrophotometer. A plateau was reached after 3 min. Using an E~~~ = 110 for oxydized DTE we calculated directly the number of disulfide b n d s present in the protein solution. I n four determinations we found an average of 3.3 0.2 mo1 of cystine per mole of inhibitor.

*

Amino Acid Analysis Four series of analyses were performed for each hydrolysis time. The results are shown in Table 5. From the concentration of each amino acid in nanomofes in the sample, it is possible to calculate the recovery of the analysis. The sample contained 113.4 mg protein; we recovered 113.5 mg. No hexosamines were found. On the basis of six half-cystines per mole, as determined by DTE (see above) the inhibitor contained 207 residues and the molecular weight calculated from these data gave a value of

22 630. A molecular weight coherent with the total amino acid composition was calculated by an iterative method due to Delaage (47). This gave a value of 22 508. Determination of N-terminal Residues The N-terminal residues of the inhibitor and of the seven peaks obtained after electrofocusing (Fig. 4A) were determined following dansylation, hydrolysis, and chromatography on polyamide sheets. In all cases leucine was found to be the only residue detectable. T o check that conclusion, Dr. David Gibson made some preliminary runs on an Illitron sequenator, according to the method described in detail in Ref. 36. The results are shown in Table 6 . Leucine was confirmed as the main N-terminal residue. However, in addition to traces of glutamic acid, glycine, and alanine there was a significant amount of aspartic acid. Qualitatively, the same results were obtained whether the analysis was performed on the inhibitor isolated directly by affinity chromatography or on two different preparations focusing at pH 6.9 in a pH gradient (see Fig. 4B and C). Characteristics of the Enzyme-Inhibitor Interaction The extent of inhibition was quantitatively estimated by the residual equilibrium activity of trypsin in the presence of a given concentration of inhibitor. It was necessary, therefore, to measure the time it took for the equilibrium to be established. Figure 6 shows that between pH 2.6 and 5.7 a constant activity of trypsin could

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ROULEAU AND LAMY: CHARACTERIZATION OF A TRYPSIN INHIBITOR

Fro. 6. ERect of incubation time on inhibition of trypsin. A solution of trypsin (274 pg active trypsin per millilitre) was prepared in a solution containing 10-3 M HC1 and 5 X M CaCB2at pH 2.6. The inhibitor purified by affinity chromatography was dissolved (228 pg/ml) in 0.05 M citrate buffer at pH 3.50, 4.73, and 5.82. In addition, a similar inhibitor solution was prepared in the HC1-CaC12 solution. At time zero, equal volumes of trypsin and of the various inhibitor solutions were mixed together at room temperature. The final pH's were 2.60,3.45,4.63, and 5.72. At various times thereafter, 200 pP of the mixt~~res were assayed at pH 8.0 according to the method of Schwert and Takenaka (17), (A-A) pH 2.60; (A-A) pH 3.45; ( 9 - 9 ) pH 4.63; (m-4) 5.72.

be measured after 2 min of contact between the inhibitor and the trypsin. The same was true up to a pH of 8.5. In this work all assays of residual trypsin activity were performed after 10 min incubation. Figure 4 suggests also that the pH of the incubation mixture influences strongly the interaction between inhibitor and enzyme. Figure 7 shows the residual trypsin activity after 10 min incubation with a constant amount of inhibitor, at different pH's. Maximum inhibition occurred between pH 7.5 and 8.0. It must be pointed out that, while the incubation took place at a variable pH, the enzymatic assay was always at pH 8.0. Specificity o/ the Inhi bition Figure 8 shows the residual activity of trypsin, chymotrypsin, and elastase after these enzymes were incubated with variable amounts of inhibitor. One milligram of protein blocked the activity of 1.09 mg of trypsin, 0.24 mg of chymotrypsin, and 0.21 n ~ gof elastase. While the antitrypsin activity was increased over that present in the initial acidic extract, the antichymotrypsin and

antielastase activities were not. From the data represented in Fig. 8 (left panel) a molecular weight of 21 300 was calculated for the inhibitor (see Table 3 ) . Determination sf K, Figure 9 shows the amount of free active trypsin, in equilibrium with increasing amounts of inhibitor. In this experiment we titrated directly the free active sites of the enzyme using NPGB as titrant. With the experimental points shown on the figure a value of K i= $.45 X M was calculated by the iterative curvefitting technique described in Methods'. This value is to be compared with that obtained following application-of the classical treatment of Green and Work (40) which was K,= 8.0 X M. Data shown on Fig. 9 permits the calculation that 1 mg of inhibitor blocked 1.13 mg of active trypsin. From this, a molecular weight of 20 560 was calculated (Table 3).

Discussion In this paper we describe the isolation of a trypsin inhibitor from acetone powder and its

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CAN. J . BBOCAEM. VOE. 53, 1975

pH of incubation mixture FIG.7. Residual activity sf trypsin at pH 8.0 after incubation with the inhibitor at various pH's. The experiment was carried out essentiaIly as described in the legend of Fig. 6 with a constant incubation time of 10 anin. The bufTers used were 0.05 M citrate between pH 3.0 and 6.0, 0.05 Tris-maleate buffer between pH 6.0 and 8.0, 8.05 M Tris buffer for pH higher than 8.0. The two different symbols represent two separate experiments. In each assay there were (a)15.8 pg active trypsin and 9.0 pg inhibitor, (A)14.5 pg active trypsin and 8.5 pg inhibitor.

inhibitor ( ~ g ) Fro. 8. Specificity of the trypsin inhibitor. The residual activities of trypsin, chymotrypsin, and elastase were measured, as described in 'Methods'. The concentrations indicated on the axis are those existing in the assay mixture.

purification by affinity chromatography. In a preliminary step the phenolic compoarnds were eliminated by extracting the fresh potato

pulp with cold acetone. The acidic and alkaline extracts of this acetone powder, after lyophilization, gave a white powder. The acetone-water

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ROULEAU AND LAMY: CHARACTERIZATION OF A TRYPSIN INHlBlTBR

inhibitor ( ~ g ) FIG.9. Determination of Ki.The stock solution of trypsin (2.57 .X 1 0-4 M) was made up in 0.012 IV HCl - 0.01 A4 CaC12. The stock solution of the inhibitor contained 3.75 mg/ml. It was prepared in a Tris b u t k 0.01 M, pH 8.2, made 0.02 M in CaC12.To 0.02 ml of trypsin solution were added variable volumnes of the inhibitor solution (0.0200.800 ml). The volumes sf the reaction mixtures were made up to 1 ml with the Tris bufier. After incubating for 5 min, the titration was started by adding 10 of a 0.01 M NPGB solution to the experimental cuvette and to the blank containing 1 ml of Tris buffer. The reaction was followed in a Cary spectrophotometer 118-C at 410 nm. The value of A,,, at t O is proportional to the amount of the free active sites of trypsin in equilibrium with the inhibitor. It was obtained by extrapolation after fitting the experimental curve to a quadratic equation. These various extrapolated values were converted to molarities of active trypsin using a value of 16 598 (21) for the molar extinction coefficient of p-nitrophenol.

-

mixture which was formed when equal volumes of acetone and potato pulp (75% water) were mixed in the first step of the preparation, must have extracted many proteins. Stegelnan et al. (48) obtained more than twenty bands on PAGE4 in freshIy prepared potato juice, while our extracts showed only six bands. The main purification step was an afinity chromatography on a Sepharose-trypsin column at pH 4.5. The protein obtained gave one band on PAGE at acidic and alkaline pH, which was not present in any of the other fractions. In spite of the fact that the inhibitor binds much more tightly at pH 8.0 (see Fig. 7), the acidic pH was selected because a monodisperse protein was obtained under these conditions. On the other hand the product obtained after affinity chroma-

tography at pH 8 showed several bands. The low affinity of the inhibitor for Sepharosetrypsin at pH 4.5 was probably responsible for the presence of inhibitory activity in the fractions eluted by the acetate buffer and NaCl (see Fig. 1 and Table 2). The molecular weight was measured by sedimentation equilibrium technique and f sund to be 20 300 and 23 600 in neutral solution of NaCl with or without 0.29 M mercaptoethanol, 19 420 and 22 130 in 6.0 M guanidinium chloride with or without mercaptoethanol, and 20 700 in 0.1 M acetic acid. In NaC1 the protein appeared monodisperse while in acetic acid the curve of In C vs. r2 was distinctly curved. However, this curvature seemed to be due to non-ideality rather than paucidispersity, since the plot of the

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972

CAN. J.

BIOCHEM.

data according to the equation of Rowe and Rowe gave a straight line.- he purified inhibitor behaved as monomeric protein since no decrease of molecular weight was observed ei,ther in guanidine chloride, mercaptoethanol, or acetic acid. Based on a molecular weight of 21 000 and on a spectrophotometric determination of disulfide bonds, t h e amino acid analysis led to a calculated molecular weight at 22 500 in agreement with the value determined by ultracentrifugation. No free SH groups, sugar, or hexosamine were found. ~ a c molecule h contained three S-S bonds and two tryptophan residues. The association of the inhibitor with trypsin was found to be rapid (-5 min) and to be maximum at a pH of 7.0 to 8.0. One milligram of inhibitor combined with 1.09 mg of active trypsin (with BAEE as substrate) and 1.13 mg trypsin (when titrated with NPGB). If a 1: 1 association is assumed, this led to molecular weights of 21 300 and 20 600, respectively. A K iof 8.5 X lo-? M was calculated. This is close to the values of the K , of trypsin toward BAEE and NPGB, and a competition between these substrates can be suspected. Thus, essentially the same molecular weight was obtained by ultracentrifugation, inhibition experiments, and amino acid analysis. Coupled with the fact that only one band was detected by PAGE at two different pH's we could conclude that the inhibitor was monodisperse and that all the molecules present in the preparation had inhibitory activity against trypsin. In spite of these indications of purity, other experiments indicated that we weredealing with a paucidisperse system. Electrofocusing experiments, in a pH gradient 3-10, separated a main component having an isoelectric point of 6.9 and, in small concentration, a variable number of species. All the fractions proved to have antitripsin activity. They were probably not products of association since reelectrofocusing of the peak at pH 6.9 gave essentially a single component with a p l of 6.9 (Fig. 4B). The N-terminal group determined by dansylation was leucine, for the inhibitor obtained by affinity chromatography as well as for all the proteins separated by electrofocusing. However, preliminary sequence determinations (see Table

VOL. 53,

1975

6) showed that, in addition to leucine, aspartic acid was present in the chromatographed inhibitor (Leu/Asp = 2/1) as well as in the protein of pl6.9 (Leu/Asp = 4 / 1) . When the inhibitor was allowed to interact with chymotrypsin and elastase (Fig. 8) we found that 1 mg inhibited about 0.2 mg of each of these enzymes. If the purified inhibitor were monodisperse and polyvalent, in absence of competition with the substrate a 1: 1 weight ratio should be observed with these enzymes as well as with trypsin. Therefore, it seemed that 20% of the molecules present in the inhibitor could combine with chymotrypsin and elastase, as well as with trypsin. In the inhibitor preparation (pl = 6.9) we found one leucine and one aspartic acid per mole of inhibitor as N-terminal residues. This could indicate that the intact inhibitor was partially hydrolyzed at its active site during affinity chromatography at pH 4.5 or that the preparation was contaminated by a polyvalent inhibitor. If aspartic acid were ,the new N-terminal group of the split inhibitor, treatment wi,th mercaptoethanol and guanidinium chloride should give two fragments with a much lowered average molecular weight. This was not observed to be the case in the ultracentrifuge. However, if the reaction site were located toward one of the ends of the polypeptide chain, it is possible that we could not detect the slight decrease in molecular weight. If the presence of two N-terminal amino acid residues were an indication of paucidispersity, then the contaminant must have had the same molecular weight and the same charge density as the main trypsin inhibitor. It must also be emphasized that proteolysis can occur when inhibitors are prepared by affinity chromatography, giving rise to new N- and Cterminal amino acids (49). It is therefore difficult to interpret the real significance of these data. Three other laboratories have purified and partially characterized potato trypsin inhibitors. The properties described differ markedly from those we report in this paper. For example, the molecular weight determined by inhibition, as reported by Iwasaki et al. (4) and by Hochstrasser and Werle (6) was of the order of 10 000. We have never found such a value. We must note however that we measured the con-

ROULEAU AND LAMY: CHARACTERIZATION OF A 'T'RYPSIN INHIBITOR

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centration of trypsin by active site titration, which indicated that close to of the protein was inactive. By gel filtration, both Iwasaki and Hoehstrasser determined molecular weights of the order of 20 OOQ. The N-terminal residue found by Iwasaki was alanine, while Hochstrasser found glutamic acid, alanine, and aspartie acid. Comparison of the amino acid composition following the method of Shapiro (50) revealed no real similarity between the inhibitors purified by Iwasaki, Hochstrasser, or ourselves. Wowever Belitz (5) has described a trypsin inhibitor (A,) having a p l of 6.9 with an amino acid cornposition compatible with that reported here. In general, we can say that potato trypsin inhibitors purified by different groups appear to be different proteins. Stegemann et al. (48) have recently shown that the numbers and types of proteins existing in different varieties of potatoes can be quite variable. A better comprehension of the diversity observed so far among the different potato inhibitors requires a more detailed study of the primary structure of the products isolated from different varieties grown in different geographical locations. 1. Vogel, R., Trautschold, I. & Werle, E. (1968) Natural Proteinase Znkibitors, Academic Press Inc., New York, N.Y. 2. Liener, I. E. & Kakade, M. L. (1969) in Toxic Constituants in Plant Foodstlifs (Liener, I. E., ed.), pp. 7-68, Academic Press Inc., New York, N.Y. 3. Ryan, C. A. (1973) Annu. Rev. Plant Pltysiol. 24, 173-196 4. Iwasaki, T., Kiyohara, T. & Yoshikawa, M. (1971) J. Biochem. 70, 817426 5. Belitz, H. D., Kaiser, K. P. & Santarius, K. (1971) Biwhent. Biophys. Res. Commcli?.42, 420-427 6. Hochstrasser, K. & Werle, E. (1969) Hoppe Seyler's 2.Plt~)siol. Chem. 350,897-902 7. Melville, J. C. & Ryan, C. A. (1972) J. Biol. Chem. 247, 3445-3453 8. Solyom, A., Borsy, J. & Tolnay, P. (1964) Bioclzenz. Plaartazucd. 13, 391-394 9. Porath, J. (1972) Biotechnol. Bioer~g.Sjymp. 3,145-166 10. Mojirna, Y., Moriya, H. & Moriwaki, C . (1971) J. Biockem. 69, 1019-1025 11. Ryan, C. A. (1971) Biockem. BiopIzys. Res. Commurt. 44, 1265-1270 12. Rancour, J. M. &Ryan, C . A. (1968) Arch. Bbchem. Biophv~.125, 380-383 13. Iwasaki, T., Kiyohara, T.& Yoshikawa, M. (1972) J. Biocheflm. 72, 1029-1036 14. Iwasaki, T., Kiyohara, T. & Yoshikawa, M. (1973) J. Bioclzern. 73, 1039-1048

973

15. Iwasaki, T., Kiyohara, T. & Yoshikawa, M. (1973) J. Biochem. 74, 335-340 16. Walsh, K. A. & Wilcox, P. E. (1970) in Methods in Enzymologj) (Perlmann, G. E. & Lorand, L., eds), vol. XIX, pp. 31-41, Academic Press Inc., New York, N.Y. 17. Schwert, G. W. & Takenaka, Y. (1955) Bioclzim. Biophys. Acta 16, 57CT575 18. Uram, M. & Lamy, F. (1969) Biochim. Biop/tys. Acta 194, 102-111 19. Lansing, A. I., Rosenthal, T. B., Alex, M. & Dempsey, E. W. (1952) Anat. Rec. 114, 555-576 20. Shotton, D. (1970) in Methods in Enzymology (Perlmann, G. E. & Lorand, L., d s ) , vol. XIX, pp. 113140, Academic Press Inc., New York, N.Y. 21. Chase, T. & Shaw, E. (1967) Biochetn. BiopItys. Res. Commurz. 29, 508-51 4 22. Dayhoff, M. 0. (1969) Atlas of Protein Sequertce attd Structure, National Biomedical Research Foundation, Silver Spring, Md., vol. 4, p. D-115 23. Kezdy, F. J. & Kaiser, E. T. (1970) in Methods in Enzymology (Perlmann, G. E . & Lorand, L,, eds), vol. XIX, pp. 3-20, Academic Press Inc., New! Y ork, N.Y. 24. Dayhof, M. 0. (1969) Atlas of Protein Seqi4ettce arrd Structiire, National Biomedical Research Foundation, Silver Spring, Md., vol. 4, p. D-118 25. Greene, L. J., DiCarlo, J. J., Sussman, A. J., Bartelt, D. C. & Roark, D. E. (1968) J. Biol. Clzem. 243, 1804-1815 26. Ardelt, W., Ksiezny, S. & Niedzwiecka-Namyslowska, I. (1970) Anal. Biochem. 34, 180-187 27. Kunitz, M. (1947) J. Get?.Physioj. 30, 291-310 28. Gertler, A. & Hofmann, T. (1970) Can. J . Biockenz. 48, 384-386 29. Erthingshausen, G., Adler, H. J. & Reichler, A. S. (1969) J. Chromatogr. 42, 355-366 30. Goodwin, T. W. & Morton, R. A. (1946) Biochem. J. 40, 628-632 31. Edelhoch, H. (1967) Biochetnistry 6, 1948-1 954 ~ . 76, 4331-4337 32. Boyer, P. D. (1954) J. Am. C l ~ e nSoc. 33. Iyer, K. S. & Klee, W. A. (1973) J. Biol. Chem. 248, 707-710 34. Dische, Z . (1962) in Metlzods in Carbohydrate Chemistry (Whistler, R. L., Wolfrom, M. L., BeMiller, J. N. & Shafizadeh, F., eds), vol. I, pp. 478-479, Academic Press Inc., New York, N.Y. 35. Woods, K. R. & Wang, K. T. (1967) Biocltinl. Biop l ~ j ~Acta s . 133, 369-370 36. Gibson, B. (1974) Biocllemistry 13, 2776-2785 37. Haglund, H. (1971) in Methods of Bimltemical Analysis (Glick, D., ed.), vol. 19, pp. 1-104, Wiley Interscience, New York, N.Y. 38. Maurer, H. R. (1971) Disc Electropltoresis and@eluted Techniqiies of Polyacrylutnide Gel Electropltoresis, p. 44, Walter De Gruyter & Co., Berlin, New York 39. Swank, R. T. & Munkres, K. D. (1971) Anal. Biothem. 39, 462-477 40. Green, N. M. & Work, E. (1953) Biochenz. J. 54, 347-352 41. Kassell, B. & Marciniszyn, M. B. (1971) in Proceedings of the International Research Conference on

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42. 43. 44.

45. 46.

CAN. J. BlOCHEM. VOL. 53, 1975

Proteir.lass lnlribitors (Fritz, H . & Tsckesche, W.,eds), pp. 43-46, Walter De Gruyter & Co., Berlin, New

5, 423-438 ~. 168, 47. Debage, M, (1968) BiwRi/17. B i ; ) ~ ) l t yAcbu Uork 573-575 Uphantis, D. A. (1964) Biochemistry 3, 297-317 48. Stegemann, H., Francksen, H. & Macks, V. (1973) Z. Naturforsch. 28c, 722-732 Rowe, 13. 1. & Rowe, A. 1.(1970) Biochim. Blop/~ys. 49. Fritz, H., Brey, B., Rluller, M. & Gebhardt, M. Acta 222, 647-659 (1941) in Proceedi~~gs of the hztert~afiotralResearch Cohn, E. J. & Edsall, J. %. (eds) (1943) in Proteins, Arnirro Acids and Peprids, p. 370, Reinhold, New Corfference o11 Brofebrase I~thibitors (Fritz, H. & Ysrk, N.U. Tschescke, H., eds), pp. 28-37, Walter De Gruyter & Co., Berlin, New York Ullmann, A., Soldberg, M. E., Perrin, D. & Monod, J. (1968) Biwlzerrtistry 7, 261-265 58. Shapirs, H. M. (1971) Biaclzini. Blop/zys. Actn 236, 725-738 Esornis, W.D. & Battaile, J. (1966) F/zytoc/remistr~~

Purification and characterization of a trypsin inhibitor from Solanum tuberosum.

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