[79]

BROAD-SPECIFICITY INHIBITORS FROM SEA ANEMONES

881

TABLE II DISTRIBUTION OF PROTEINASE INHIBITORS IN VARIOUS SNAKE VENOMS a'b

Family

Bovine plasma kallikrein

Bovine plasma plasmin

Bovine pancreatic trypsin

Bovine pancreatic (~-chymotrypsin

12 40 88

18 91 110

5 22 74

5 77 217

100 100 100 4 125 215

62 12 42 20 34 43

55 3 60 22 18 28

80 15 20 13 34 105

Viperidae

V. russelli V. ammodytes B. arietans Elapidae

N. N. N. H. D. D.

hannah nivea haje haemachalus angusticeps polylepis

a From H. Takahashi, S. Iwanaga, a n d T. Suzuki, Toxicon 12, 193 (1974). b The values were expressed as the quantities (#g) to show 50 % inhibition of the activity of the proteinases. The following venoms showed no inhibitory activity when 100 t~g of each was used: V. pale~tinae, E. carinatu~, B. gabon@a, N. naja

atra, N. melanoleuca, N. nigricollzs, N. naja samarens~s, N. naja, B. fasczatus, B. mult~cinctus, L. semifasciata, L. lahcaudata, A. halys blomho~,, A. acutus, A. rhodostoma, A. p~scwo~us leucostoma, A. contortmx contortr~x, A. p~scivorus piscworus, A. contortr~x mokeson, C. atrox, C. adamanteus, C. viridis wr~dis, C. basiliscus, C. durissus terrificus, B. atrox, B. jararaca, T. flavoviridis, T. gramineus, T. okinavensis, T. mucrosquamalus, Causus rhombeatus.

Distribution2 ,14 Proteinase inhibitor of the same type as described here is demonstrated also in the venoms of several snakes of the Elapidae and Viperidac (Table II). However, it is not found in Crotalidae and Hydrophiidae venoms. ~4D. J. Strydom, Nature (London) New Biol. 243, 88 (1973).

[79] B r o a d - S p e c i f i c i t y I n h i b i t o r s f r o m Sea Anemones By GERT WUNDERER, L~SZLO BI~RESS, WERNER MACHLEIDT, and HANS FRITZ In the purification of neurotoxins from Anemonia sulcata, fractions with antitryptic activity were observed. 1 This inhibitory activity could be ascribed to a variety of basic polypeptides. ~,~ The broad specificity 1 L. B6ress and R. B~ress, Kiel. Meeres]orsch. 27, 117 (1971). : H. Fritz, B. Brey, and L. B6ress, Hoppe-Seyler's Z. Physiol. Chem. 353, 19 (1972). 3G. Wunderer, K. Kummer, H. Fritz, L. B~ress, and W. Machleidt, Proteinase Inhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 277.

882

N A T U R A L L Y OCCURRING P R O T E A S E I N t t I B I T O R S

[79]

of these inhibitors for trypsin, chymotrypsin, plasmin, and kallikreins closely resembles that of the basic pancreatic trypsin inhibitor (BPTI).2 The present article describes our revised procedure for the isolation of the proteinase inhibitors from A. sulcata. Assay Method Trypsin inhibition by the sea anemones inhibitors is tested with N "benzoyl-DL-arginine p-nitroanilide (BAPA) as substrate, as described by Kassell. 4 Definition o] Units. One tryptic unit is defined as the enzyme activity that cleaves 1 ~mole of DL-BAPA per minute under the described conditions. One inhibitor unit reduces the trypsin-catalyzed hydrolysis of DLBAPA by 1 ~mole/min. Purification Procedure

Step 1. Preparation o] the Sea Anemones. Anemonia sulcata were collected in the bay of Naples. The fresh sea anemones are washed and centrifuged for 10 min at 1500 g. The supernatant is discarded because it contains only negligible amounts of inhibitors (and toxins). The remaining sediment is deep-frozen and stored at --20 °. After this procedure the dry weight of the material amounts to 13% of its wet weight. Step 2. Extraction. Two kilograms of prepared sea anemones are homogenized in 2 liters of 96% ethanol with a Cenco Waring blender in several portions. The homogenate is heated transiently to 60 °. After cooling to room temperature, the precipitated proteins and cell fragments are spun down (15 rain at 2000 g). The brown supernatant is collected. The sediment is homogenized for a second time in ] liter of 50% ethanol, heated to 50 ° , cooled, and centrifuged. This time the residue is discarded. The combined supernatants are dialyzed for 16 hr against 40 liters of deionized water and filtered through a paper disk (Seitz K2) on a 30-cm Biichner funnel. Step 3. Batchwise Adsorption o] the Inhibitors on CM-Cellulose. Batch I: The extracts obtained in step 2 are diluted with deionized water to a conductivity of 4 InS X cm-1, about 16 liters total, and adjusted to pH 6.5 with acetic acid. Then 60 g of dry CM-cellulose (Serva; capacity: 0.55 mEq/g) are added; after readjustment of the pH to 6.5, the suspension is stirred for 30 min. The suspension is filtered through a fritted glass filter, and the loaded cellulose exchanger is washed with deionized water. The CM-cellulose, batch I, contains most of the inhibitors 4 B. Kassell, t h i s series, Vol. 19, p. 845.

[79]

BROAD-SPECIFICITY INHIBITORS FROM SEA ANEMONES

883

(60.5%) and a small amount of toxin II (9% of total toxicity, cf. B~ress et al.5,6). Batch II: The filtrate of batch I is adjusted to pH 5.0 with acetic acid and diluted to a conductivity of 2 mS X cm-1. Then 60 g of dry CM-cellulose are added. After correction of pH, the suspension is stirred for 30 rain and filtered; the cellulose is washed thoroughly with deionized water. This CM-cellulose, batch II, contains the remaining inhibitors and another part of toxin II (20% of total toxicity). For the isolation of the toxins along with the inhibitors, a third batch (SP-Sephadex C-25, pH 3.2, 2 mS X cm-1) is processed. This batch III does not contain inhibitors. For the isolation of the inhibitors, the proteins adsorbed in batches I and II are eluted from CM-cellulose with about 1.5 liters of buffer (0.05 M Tris-HC1, pH 8.0, 1 M NaC1) each. The elutes are concentrated by ultrafiltration with an Amicon UM-05 or UM-2 filter to final volumes of about 70 ml. Step ~. Gel Filtration on Sephadex G-50. The concentrated solutions obtained in step 3 are each chromatographed on a 7 X 120 cm Sephadex G-50 medium column equilibrated and developed with 2% acetic acid (v/v) at a flow rate of 3 ml/min. The inhibitors are eluted in 3 to 4 times the void volume separated from accompanying proteins and colored products. The inhibitor-containing fractions are collected and concentrated in a rotary vacuum evaporator. As the second half of the inhibitor peak still contains salts, it is desalted again on Sephadex G-25 (column: 5.6 X 120 cm, equilibrated and developed with 2% acetic acid (v/v), 120 ml/hr) and finally lyophilized. The yields and purification factors calculated from steps 2-4 are summarized in Table I. Step 5. Ion-Exchange Chromatography on SP-Sephadex. The inhibitor fraction from step 4 can be separated into 10 multiple forms by ionexchange chromatography on SP-Sephadex. A microanalytical system was used to find optimal conditions for the separation. 7 This system consists of a 0.1 t 100 cm microbore column filled with SP-Sephadex C-25, equilibrated with 25 mM phosphate buffer, pH 6.13 (50 mM Na +) and developed with a concave gradient of sodium chloride in 25 mM phosphate buffer. Continuous flow-cell photometry at 224, 253, and 280 nm permits a detection of less than 1 nmole (about 7 ~g) of inhibitor in the eluates as well as the characterization of peaks by their different UV absorption. From the reproducible results obtained with this system we were able to select the most suitable sodium ion concentration range for 5 L. B~ress, R. B~ress, and G. Wunderer, FEBS Lett. 50, 311 (1975). 6 L. B~ress, R. B~ress, and G. Wunderer, Toxicon 13, 359 (1975). 7 W. Machleidt, W. Kerner, and J. Otto, Z. Anal. Chem. 252, 151 (1970).

884

N&TURALLY OCCURRING PROTEASE INHIBITORS

[70]

TABLE I YIELDS

AND

PURIFICATION IN

FACTORS THE

Step

Procedure

2

C o m b i n e d extracts dialysis E l u a t e from b a t c h I E l u a t e from b a t c h I I I n h i b i t o r fraction after Sephadex G 50: B a t c h I Batch II

3 4

CALCULATED

ISOLATION

FOR

STEPS

2,

3,

AND

4

PROCEDURE

IU a

(IU X m l ) / A 2s0b

Purification factor

Yield (%)

3750 6500 1570 1380

9700 8400 6550 1820

0.065 0.072 3.92 1.41

1 1.1 60.5 21.7

100 86.5 67.5 18.8

1570 910

6500 776

5.64 2.30

86.8 35.4

66 16.9

Volume (ml)

a Trypsin inhibitory activity, substrate: N=-benzoyl-DL-arginine p-nitroanilide; 1 I U is equivalent to the inhibition of a b o u t 1 mg of bovine trypsin. b Absorption at 280 nm.

the preparative separation of the inhibitors (Fig. 1). The same system was used to check the purity of the fractions isolated on a preparative scale. The preparative 1.5 X 100 cm SP-Sephadex C-25 column is equilibrated with a 50 mM phosphate buffer (100 mM Na+), pH 6.13, and developed with a linear gradient of NaC1 in the starting buffer up to 400 mM Na ÷ and in a second step from 400 to 1000 mM Na ÷ (flow rate 70 ml/hr; Fig. 2). The 9 inhibitor fractions obtained in this chromatography are each desalted on a 3.5 X 120 cm Sephadex G-50 superfine column (50 mM NH4HCQ pH 8.1, flow rate 50 ml/hr) and lyophilized. Step 6. Rechromatography o] the Inhibitors on SP-Sephadex. Inhib1000 o o

o c EEE ,,,...,.o.~ "-- O,lOw,{.~l

g°

° °

0 :

50

i!~

~"

.

"6

'

'

~ '

2 3

h

I

1

~ \

--o

1 0 0 F o ~ ...............'

I

1 ~ ";

" so'

:" ""o

: '

1 /

5

6

ol ~.

/

-500

o

]

°l ?

i ":

.......~

~

:

"

%

~

~

'

'

,oo'

'

'

;': [ \

i \ .'.

Z

I '

,sb

E °°

Tube number

FIG. 1. Separation of proteinase inhibitors from Anemonia sulcata. Analytical scale: 0.1 × 100 cm SP-Sephadex C-25 column equilibrated with 25 m M phosphate buffer, p H 6.13, 56 m M N a ÷, and developed with a concave NaC1 gradient (up to 1 M Na+), 0.385 ml/hr, 2 tubes/hr, 25 °. 250 nmoles of the inhibitor mixture were applied. Transmissions were registered continuously at 224 ( . . . . . ), 253 ( . . . . ), and 280 ( ~ ) nm.

[79]

BROAD-SPECIFICITY INHIBITORS FROM SEA ANEMONES

885

i.0]

8 7oo•

~

•

N

500

•

~

•

B

• . '

.

,

•

300 2

Nil 0 Tube number

FIG. 2. Preparative separation of proteinase inhibitors on a 1.5 X 100 cm SPSephadex C-25 colum equilibrated with 50 m M phosphate buffer, p H 6.13, 100 m M Na ÷, and developed first with a linear NaC1 gradient up to 400 m M Na ÷ followed by a second linear NaCI gradient from 400 to 1000 m M Na ÷, flow rate 70 ml/hr, 6 tubes/hr, 25°. Up to 1 g of the inhibitor mixture was applied. Upper boldface line:

absorption at 280 nm- thin line: inhibitor activity in IU. itor fractions 2, 3, 5I, 5II, 6, 7, and 8 (cf. Fig. 2) are further purified by chromatography on a SP-Sephadex C-25 column (1 X 100 cm, flow rate 50 ml/hr). For this purpose the column is equilibrated with 50 m M phosphate buffer pH 6.13 and developed in equilibrium with selected Na ÷ concentrations. The yields of the inhibitors obtained in step 5 and the conditions for rechromatography (step 6) are given in Table II. The rechromatographed inhibitors are desalted on Sephadex G-50 as described in step 5 and lyophilized. Inhibitor I is further purified by affinity chromatography on trypsin covalently b o u n d to CM-cellulose as described earlier.2, s Properties

Stability. Like other low-molecular-weight trypsin inhibitors, the inhibitors from Anemonia sulcata are very stable. They do not loose activs G. Wunderer, H. Fritz, W. Br(immer, N. Hennrich, and H.-D. Orth, Biochem. Soc. Trans. 2, 1324 (1974).

886

NATURALLY OCCURRING PROTEASE INHIBITORS

[79]

TABLE II RELATIVE YIELDS IN SP-SEPHADEX CHROMATOGRAPHY (STEP 5) AND CONDITIONS FOR RECHROMATOGRAPHY (STEP 6)

Inhibitors

Relative yields BatchI (%) in step 5 BatchlI (cf. Fig. 2) Na + concentration, used in reehromatography (mM) (Step 6)

1

2

3

4

5

6

7

8

9

2.4 2.3

5.2 5.2

14.0 14.0

18.5 21.0

24.4 29.0

4.8 3.0

3.2 1.6

2.8 0.4

0.5 0.2

--~

205

205

__b

330

350 470 510

__b

Enriched by affinity chromatography [H. Fritz, B. Brey, and L. B~ress, HoppeSeyler's Z. Physiol. Chem. 353, 19 (1971); G. Wunderer, H. Fritz, M. Brtimmer, N. Hennrich, and H.-D. Orth, Biochem. Soc. Trans. 2, 1324 (1974)]. b Not rechromatographed. ity when incubated at room t e m p e r a t u r e in the p H range 1.5 to 10 for several hours. Lyophilized material stored at - - 2 0 ° retains its activity for at least 3 years. Molecular Weight. As estimated by gel filtration on calibrated Sephadex G-50 columns, the isolated inhibitors have molecular weights between 5500 and 7000. These values are confirmed by SDS gel electrophoresis of inhibitor 5 I I in its reduced form. T h e y are also in agreement with those values calculated from the specific trypsin inhibitory activity. Amino Acid Compositions. T a b l e I I I gives the amino acid compositions of inhibitors 1 to 7 as calculated for molecular weights between 5500 and 7000. Inhibitor 5 I I (the m a j o r component) is composed of 60 residues and has a calculated molecular weight of 6799.8. Its molecular absorption coefficient is AlV, --1 cm at 280 nm = 7.24. N-Terminal Residues. With the dansylation technique, the N - t e r m i nal amino acid residues of inhibitors 1-5 I I were found to be isoleucine, whereas for inhibitors 6-9 an end group could not be detected. In manual E d m a n degradation of inhibitor 5 I I , isoleucine was found as a single N terminal residue, in 81% yield, followed by asparagine and glycine. Specificity. Each of the isolated inhibitors inhibits strongly bovine trypsin and chymotrypsin, porcine plasmin, and porcine pancreatic kallikrein. 2,3 H u m a n and porcine serum kallikreins, 9 h u m a n plasmin, TM as 9 G. Wunderer, K. Kummer, and H. Fritz, Hoppe-Seyler's Z. Physiol. Chem. 353, 1646 (1972). ~oW. Kalckreuth, Dissertation, Faculty of Medicine, University of Munich, 1972.

[79]

BROAD-SPECIFICITY INHIBITORS FROM SEA ANEMONES

887

TABLE III AMINO ACID ANALYSIS DATAa FOR INHIBITORS ] TO 7 (MOLES PER MOLE) Residue Asp TAr Ser Glu Pro Gly Ala Cys b Val Mct Ile Leu Tyr Phe His Lys Arg

1

2

3

4

5I

5 II

6

7

6.00 1.91 4.71 4.07 3.72 8.27 2.15 5.98 2.09 -1.84 2.87 2.16 1.39 1.98 4.46 4.98

6.00 1.75 3.80 4.95 2.88 6.73 2.05 5.05 2.14 -0.92 1.89 3.43 2.37 0.99 3.70 4.67

6.00 1.00 4.32 5.06 2.75 6.11 1.59 5.82 3.17 -1.62 2.13 3.17 2.08 1.78 4.00 5.72

6.00 1.79 4.11 4.89 2.89 7.21 2.75 2.82 3.05 0.30 1.69 2.53 2.60 2.64 0.83 4.10 4.66

6.14 1.03 3.55 5.01 2.64 7.00 2.06 5.79 2.39 -1.47 2.11 2.36 2.98 1.07 3.72 6.39

6.00 0.99 3.94 5.00 2.18 6.95 1.75 5.95 3.92 -1.82 2.86 3.94 2.99 1.00 3.76 6.31

6.34 1.40 3.83 4.06 2.11 7.00 2.67 5.31 2.82 -1.00 2.83 2.07 2.46 1.56 3.87 5.90

6.00 3.45 3.62 2.24 2.90 6.29 6.98 5.30 2.63 0.85 0.48 1.22 2.07 1.79 1.07 6.85 2.00

a The values given were obtained from 20-hr hydrolyzate and are not corrected. b Determined as cysteic acid after performic acid oxidation. well as boar sperm acrosin, 11 are also i n h i b i t e d b y the m i x t u r e of the m u l t i p l e i n h i b i t o r forms. Kinetic Properties. T r y p s i n is i n h i b i t e d s t o i c h i o m e t r i c a l l y (molar r a t i o 1:1) b y i n h i b i t o r 5 II. T h e dissociation c o n s t a n t of the complex of i n h i b i t o r 5 I I with b o v i n e t r y p s i n is d e t e r m i n e d to a p p r o x i m a t e l y 3 X 10 -1° moles per liter according to Green a n d Work. 12 T h e affinities of the Anemonia sulcata i n h i b i t o r s a n d of B P T I to t r y p s i n , p l a s m i n , a n d c h y m o t r y p s i n are comparable2; the affinities to porcine p a n c r e a t i c k a l l i krein, however, increase with the b a s i c i t y of the i n h i b i t o r s 2 I n c o n t r a s t to our results p u b l i s h e d earlier, 3 we could n e i t h e r degrade nor m o d i f y i n h i b i t o r 5 I I with T P C K - t r e a t e d t r y p s i n from E. Merck, D a r m s t a d t , u n d e r v a r i o u s acidic conditions (4 m o l e / 1 0 0 moles t r y p s i n ; p H 3.0, 3.75, 4.0, a n d 5.0). This d i s c r e p a n c y m a y be explained b y the action of accomp a n y i n g proteinases in the t r y p s i n p r e p a r a t i o n used formerly.

Discussion T h e procedure described p e r m i t s the isolation of 10 p r o t e i n a s e i n h i b itors from Anemonia sulcata. I t proved to be r e p r o d u c i b l e in at least 20 11H. Fritz, B. FLrg-Brey, H. Schiessler, M. Arhnold, and E. Fink, Hoppe-Seyler's Z. Physiol. Chem. 353, 1010 (1972). 1.. N. M. Green and E. Work, Biochem. J. 54, 347 (1953).

888

NATURALLY OCCURRING PROTEASE INHIBITORS

[79[

independent preparations. The procedure can be used as a general method for the separation of basic and neutral polypeptides from tissues. For example, using this procedure 3 neurotoxins from A. sulcata 5,6 and 4 neurotoxins from C o n d y l a c t i s aurantiaca 1"~ could be separated from the more basic inhibitors. For the isolation of all basic and neutral polypeptides, we recommend that the complete procedure, including 3 batches, be foN lowed. If only the proteinase inhibitors from A. sulcata are to be isolated, a single batch II is sufficient. As judged from chromatographic data, amino acid compositions, and N-terminal end-group determinations, at least inhibitors 2, 3, 5 I, 5 II, and 6 are pure polypeptides. Inhibitor 5 II was also shown to be pure using SDS gel electrophoresis. The amino acid compositions found for the other inhibitors suggest the presence of minor impurities. A further purification of these fractions was not attempted. A comparison of t h e amino acid compositions shows that the inhibitors from A n e m o n i a sulcata differ only in minor amino acid exchanges, i.e., they are very probably isoinhibitors. The main inhibitor fraction 5 II was chosen for structural investigations. 14,1~ From its amino acid composition, a homology with the known sequences of B P T I , 16 CTI, 1~ and inhibitors from snails, TM and vipers TM is to be expected. This is supported by preliminary results of the sequence determination24,~5 The N-terminal sequence of inhibitor 5 II, Ile-Asn-GlyAsp-Cys-Glu-Leu-Pro-Lys-Val-Val-Gly-Pro-Cys-Arg-Ala-Arg-Phe-ProArg- T y r - T y r - T y r - A s n - S e r - S e r - S e r - L y s - A r g - C y s - G l x - L y s - P h e - I l e - T y r Gly-Gly-Cys-Arg- is homologous with the sequence in positions 5-39 (which includes the active center) of B P T I , and there is a further sequence Lys-Val-Cys-Gly-Val-Arg-Ser in inhibitor 5 II which is partially homologous with positions 53-56 of B P T I . Therefore we confirm that the inhibitor 5 II from sea anemones contains Arg in its reactive center as postulated earlier. 2 29R. B6ress, L. B6ress, and G. Wunderer, Hoppe Seyler's Z. Physiol. Chem. 357, 409 (1976). ~4G. Wunderer, L. B6ress, W. Machleidt, and H. Fritz in "Protides of the Biological Fluids, 23rd Colloquium" (H. Peeters, ed.), p. 285. Pergamon, Oxford and New York, 1976. ~5G. Wunderer, Dissertation, Technical University of Munich, 1975. ~ B. Kassell and M. Laskowski, Sr., Biochem. Biophys. Res. Commun. 18, 255 (1965). ~ D. ~echov~, V. Jon~kov£, and F. ~orm, Proteinase Inhibitors, Proc. Int. Res. Con]., 1st, Munich, 1970, p. 105. de Gruyter, Berlin, 1971. ~ T. Dietl and H. Tschesche, Proteinase Inhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V) Grosse Ledder, 1973, p. 254. Springer-Verlag, Berlin and New York, 1974. ~9H. Takahashi, S. Iwanaga, Y. Hokama, T. Suzuki, and T. Kitagawa, FEBS Lett. 38, 217 (1974).