Metabolism in marine flatfish—III. Measurement of elastase activity in the digestive tract of dover sole (Solea solea L.)

Comp. Biochem. Physiol. Vol. 81B, No. 3, pp. 695-700, 1985

0305-0491/85 53.00 + 0.00 ~5 1985 Pergamon Press Ltd

Printed in Great Britain

M E T A B O L I S M IN M A R I N E F L A T F I S H - - I I I . M E A S U R E M E N T OF ELASTASE ACTIVITY IN THE DIGESTIVE T R A C T OF D O V E R SOLE (SOLEA SOLEA L.) J. C L A R K , N. L. MACDONALD and J. R. STARK Department of Brewing and Biological Sciences, Heriot-Watt University, Edinburgh EHI 1HX, UK. (Tel: 031-225-8432)

(Received 21 November 1984)

Abstract--l. A critical assessment of different methods for measuring elastase activity in crude preparations has been made using whole intestinal homogenates of Dover sole. 2. The use of the natural substrate elastin or its dyed derivatives gave optimal pH values in the alkaline region (pH 9.4-9.8) whereas artificial substrates showed optimal hydrolysis nearer neutrality in the region pH 8.1-8.2. 3. Exoproteases may interfere with certain assay procedures. 4. The properties of Dover sole elastase have been further investigated using chromatographic techniques which indicated that the main elastase activity has a molecular weight of approximately 19,500 and an isoelectric point in the region of pH 5.7.

INTRODUCTION

Elastase in fish

The first report of elastase as a distinct and separate vertebrate pancreatic enzyme was that of Balo and Banga (1950) who later isolated and semi-purified this enzyme from bovine pancreas. Subsequently elastase has been isolated and purified from the pancreatic tissues of many other vertebrates. Although able to digest a wide range of proteins including haemoglobin, casein, fibrin and denatured collagen (Mandl, 1962) pancreatic elastase is characterized by its ability to solubilize cross-linked elastin protein fibres present in connective tissue. Solubilization of elastin has been associated with a change in molecular state from rod-shaped to globular particles (Partridge et al., 1955). Elastin fibres are known to consist of a large proportion of neutral amino acids with aliphatic side-chains and preferential hydrolysis of cross-linking peptide bonds involving such amino acids is suggested as a reason for elastase substrate specificity (Shotton, 1970). Studies on the hydrolysis of synthetic ester substrates (Kaplan and Dugas, 1969) by purified porcine elastase have shown the affinity of this enzyme for the cleavage of N-benzoyl-e-alanine methyl ester (Bz-Ala-O-Me). Later experiments by Geneste and Bender (1970) used N-benzyloxycarbonyl-L-alanine p-nitrophenyl ester (N-CBZ-L-Ala-pNPE) and Nfurylacroyl-e-alanine methyl ester as specific elastase substrates. However it was stated that spontaneous hydrolysis of the p N P ester at high pH values limited its use in the determination of the pH dependence of enzyme activity. Gertler and Hofmann (1970) have suggested N-acetyl-e-alanine-e-alanyl-L-alanyl methyl ester [Ac(Ala)3-OMe] as a useful elastase substrate resistant to both trypsin and chymotrypsin attack while Bieth et al. (1974) state that N-succinylL-alanyl-L-alanyI-L-alanine-p-nitroanilide[Suc(Ala)3NA] is a highly sensitive substrate for the measurement of elastase activity in crude biological extracts.

The first report of elastase in fish was that of Lansing et al. (1953) who recorded high elastolytic activity in pancreatic islet tissue extracts of the teleost angler or monkfish, Lophius piscatorius. More recently, Zendzian and Barnard (1967) have recorded elastase-like activity in extracts of pancreas and pyloric caeca of the elasmobranch stingray, Dasyatis americana and teleost tuna, Thunnus secundodorsalis respectively. Nilsson and Fange (1969) showed elastase activity to be present in intestinal juice and pancreatic extract of the holocephalian, Chimaera monstrosa while de Haen and Gertler (1974) isolated and purified two elastase-like enzymes from pancreas of the African lungfish, Protopterus aethiopicus. In more recent work Yoshinaka et al. (1982) examined a purified elastase-like enzyme from the pancreas of the catfish, Paracilurus asotus. There have been few reports of ester substrates being used to measure elastase activity in fish extracts, de Haen and Gertler (1974) recorded the hydrolysis of Ac(Ala)3-O-Me by two elastases purified from the African lungfish, while Yoshinaka et al. (1982) recorded activity of both crude extracts and purified enzyme using Suc(Ala)3NA. The most common methods for measuring elastase activity in fish extracts involve the solubilisation of dyed elastin substrates. Zendzian and Barnard (1967) recorded enzyme activity in crude extracts using the orcein-elastin method of Sachar et al. (1955) while de Haen and Gertler (1974) and Yoshinaka et al. (1982) used this method for assaying purified enzyme from catfish. The Congo-red-elastin method of Naughton and Sanger (1960) was employed by Nilsson and Fange (1969) on crude extracts of pancreas and intestinal mucosa. Mandl (1962) states that the former method is more reliable since orcein is stainspecific for elastin and not affected by other connective tissue protein impurities such as collagen.

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M e a s u r e m e n t o f soluble protein released by enzyme activity on native elastin as used by Lansing et al. (1953) is a m e t h o d also subject to errors due to substrate protein impurities. Elastase enzymes previously purified from vertebrates are k n o w n to exist in zymogen form as proetastase, which can be activated by pancreatic trypsin (Shotton, 1970; Hartley a n d Shotton, 1971). Similar activation of fish elastase in crude extracts by trypsin has been reported by Nilsson a n d F a n g e (1969) while de H a e n a n d Gertler (1974) a n d Y o s h i n a k a et al. (1982) n o t e d that p r e i n c u b a t i o n o f the purified enzyme with bovine trypsin increased overall activity towards synthetic ester substrates. It has also been d e m o n s t r a t e d t h a t elastase from the catfish ( P a r a cilurus asotus) exists in two forms ( Y o s h i n a k a et al., 1982), t h a t one of these enzymes is a serine proteinase ( Y o s h i n a k a et al., 1983) a n d the other is a metalloproteinase ( Y o s h i n a k a et al., 1984).

discarded and the preparation dried under vacuum at room temperature. Enzyme extracts were prepared by stirring the powder with water or buffer for 2 hr at 5°C. The mixtures were then homogenised in a glass-glass homogenizer and the supernatant solution removed by centrifugation to give a 1:70 extract, which was generally lyophilised and stored at 0°C. An alternative procedure which was sometimes used was to concentrate the aqueous extract from the powder using an Amicon Ultrafiltration unit with a YMS membrane (cut off at mol. wt 5000). This was carried out in a Model 202 cell at 5C. Substrates and chemicals The majority of chemicals were supplied by Sigma Chemical Co. Ethylene diamine tetraacetic acid (EDTA) and N-ethylmaleimide (NEM) were supplied by British Drug Houses Ltd. Column packings for chromatofocusing, gel filtration were supplied by Pharmacia UK Ltd. and ionexchange material was supplied by Whatman, Chemical Separation Ltd., UK. Enzyme assays

Proteases in Dover sole

In previous investigations in this series, the c a r b o h y d r a s e s a n d the proteases in D o v e r sole were examined (Clark et al., 1984, 1985) respectively. In this latter paper, three regions o f proteolytic activity were identified. A n acidic region at pH 1-2 containing a pepsin-like protease, a neutral region corres p o n d i n g to trypsin, c h y m o t r y p s i n a n d collagenaselike enzymes and, in addition, an alkaline region of activity at p H 9.5-10.5 in which a n elastase-like enzyme a p p e a r e d to act. A l t h o u g h the p H of the fish intestine is p r o b a b l y m a i n t a i n e d in the " n e u t r a l " region, the alkaline activity in D o v e r sole intestinal extracts is, nevertheless, quantitatively the strongest as m e a s u r e d by the casein method. The purpose o f this work was to investigate the alkaline region a n d the elastase activity. MATERIALS AND METHODS

Fish

Juvenile and adult sole samples were supplied by Seafresh Farms (Scotland) Ltd., Hunterston. 0-group juveniles weighed between 15-34 g and were 50-150 mm long whereas adult fish were 2-group (average weight 400 g and length 300 ram). All fish had been continuously fed on artificial diets for at least 3 weeks (so there were no contaminating enzymes from live diets in any of the preparations). Preparation o f extracts The majority of these studies were carried out on juvenile fish using homogenates of the whole digestive tract. For examination of different parts of the intestine the alimentary canal of adult fish was divided into stomach, foregut, midgut, hindgut and rectum. Due to a lack of regional differentiation such divisions were arbitrary, being made on the basis of percentage of total gut length as suggested by Braber and de Groot (1973). All fish were starved for 3 hr before being killed by a blow to the head followed by deep-freezing. After partial thawing the intestine could be readily excised, weighed and homogenized with distilled water to give a 1: 10 homogenate. Dilutions were carried out as required. The lumen contents, present in the gut, were included in all homogenates unless otherwise stated. Acetone or butanol powders were prepared by macerating whole intestines from adult Dover sole for 10 min in ice-cold solvent (2 vol) using a Sorvall bench Omni-mix. This was followed by three extractions by stirring with the solvent at 5°C. The mixture was centrifuged, the bulk of the solvent

The buffers used in all enzyme assay procedures were 0.1 M HCI (pH 1.0), glycine-HC1 (pH 1.0-1.7), citratephosphate (pH 1.7-7.8), glycine-NaOH (pH 7.8--10.1) and phosphate-NaOH (pH 10.1-11.2). All enzyme assays were carried out at 37°C. Total proteolytic activity was estimated by a modification of the casein hydrolysis method of Kunitz (1947). Digests consisted of 1~o casein (0.5ml), intestinal homogenate (0.25 ml) and buffer (0.25 ml). After incubation, ice-cold 53/, trichloroacetic acid (1.5 ml) was added and the mixture left at 2"C for 30min. The samples were then centrifuged at 2000g for 10min and the absorbance of the supernatant solution recorded at 280 nm. Tyrosine was used as standard and the units expressed as #g tyrosine liberated/min/g protein. The protein content of the homogenate was determined by the method of Lowry et al. (1951) as modified by Miller (1959). In addition to the assay of alkaline protease activity at pH 10.1 using the above method, elastase activity was also measured routinely using elastin-orcein (Sachar et al., 1955) by a modification of the method described in the Worthington Enzyme Manual (1972). Elastin-orcein (10 mg) was continuously rotated with buffer (1.0ml) and enzyme extract (0.1 ml) for 1 hr. The mixture was centrifuged and the absorbance read at 570 nm against the appropriate elastinorcein blank which had been incubated for the same time at the same pH. Congo-red-elastin was used as a substrate in a similar manner to that described for elastin-orcein except that the absorbance was read at 495 nm after 2 hr incubation period and centrifugation. The natural substrate elastin was also examined. In the first of these methods elastin (10mg) was incubated with buffer (1 ml) and a 1:20 homogenate (0.1 ml) for 2hr. Icecold 5~o trichloroacetic acid (1.5 ml) was added to precipitate protein and, after centrifuging at 3000g for 10 min, a sample (1 ml) was taken for analysis by the Lowry method. In the second procedure the incubation and precipitation were carried out as above but the final analysis was using a sample (0.5ml) diluted to 2ml before estimating the ~t-amino nitrogen by the ninhydrin method. Hydrolysis of N-succinyl-e-alanyl-e-alanyl-L-alanine pnitroanilide [Suc(Ala)3NA] was followed by a modification of the method of Beith et al. (1974). A 4.5~o solution of the substrate (20/~1) was continuously shaken with a 1: 10-homogenate (0.25ml) and buffer (2.25 ml) for 4 hr. The mixture was centrifuged and the absorbance of the supernatant solution read at 410 nm. Assays with N-benzoxycarbonyl e-alanine p-nitrophenyl ester (N-CBZ-L-AIa-pNPE) (Geneste and Bender, 1970) were carried out using a 3 mg/ml suspension of the substrate (0.5ml) incubated with a 1:40 homogenate (0.5 ml) and

Metabolism in marine flatfish--III buffer (0.5ml) for 40min. To an aliquot (1 ml) of the supernatant solution obtained after centrifugation, 0.1 M-potassium bicarbonate solution (0.4 ml) was added and the absorbance recorded at 420 nm against the appropriate blank. Inhibitor studies were carried out by pre-incubating a 1:40 intestinal homogenate with inhibitor for 1 hr at room temperature (19°C) before assaying for residual activity using the casein hydrolysis method previously described. These experiments were carried out at three pH values (6.9, 8.2 and 10.0) and the corresponding activities were also measured in the absence of inhibitors.

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Gel permeation chromatography was carried out to determine the molecular weight and the isoelectric point and partial purification were examined by chromatofocusing (Sluyterman and Elgersma, 1978). Molecular weight determination was carried out using the partially purified material prepared by the application of a 9.6 ml sample of butanol treated extract on to a Whatman CM52 cation exchange column (1.5 x 20cm). Fractions (5 ml) were collected and eluted with buffer (0.5 mM Pipes NaOH, pH 7.0) using a salt gradient (0-0.3 M NaCI). The column was finally eluted using 0.5 M NaCI in the elution buffer. Fractions (52-90) containing high proteolytic activity towards casein at pH 10.0, orcein-elastin and Congo redelastin were combined and freeze-dried. The sample of freeze-dried extract (26.4mg protein) dissolved in water (6ml) was applied to a column of Sephadex G200 (1.5 x 90cm). The column was eluted with water at a flow rate of 8.6 ml/hr and fractions (5 ml) collected. A similar enzyme solution (6 ml) was applied to a column of PBE 94 (1.0 x 30cm) which had been equilibrated with starting buffer (imidazole-HC1 pH 7.4). The column was then eluted with "'Polybuffer 74" and fractions (6 ml) collected. The flow rate was controlled by a peristaltic pump at 22.8 ml/hr and the pH gradient ranged from 7.4 to 4.0. Finally, the column was cleared using 1 M sodium chloride in "Polybuffer 74". All chromatographic procedures were carried out at 5°C and fractions were stored at 0°C.

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RESULTS The pH-profile o b t a i n e d using casein as substrate (Fig. la) showed a high level o f activity at p H 9.5-10.5. Since this was not a t t r i b u t a b l e to trypsin, c h r o m o t r y p s i n or collagenase-like activities which all h a d optimal activites in the region p H 7-8, elastase was considered as a possible c o n t r i b u t o r to this activity (Clark et aL, 1985). The use of elastinorcein as substrate indicated t h a t at least part of this alkaline activity towards casein was a t t r i b u t a b l e to elastase' (optimal activity at p H 9 . 8 ) . This was s u b s t a n t i a t e d using Congo-red-elastin which gave an o p t i m u m value at p H 9.4 (Fig. lb). W h e n elastin itself was used as substrate the o p t i m u m p H d e p e n d e d on the assay procedure used. With the Lowry assay, which measures the a m o u n t o f phenolic a m i n o acid in the s u p e r n a t a n t solution a n d therefore gives a n estimate o f the a m o u n t o f peptides released by the enzyme action, the o p t i m u m p H value was 9.5 (Fig. 2). However, if the solubilized p r o d u c t s were estim a t e d using ninhydrin, which measures c~-amino nitrogen then the value was 8.3. The artificial substrates Suc(AIa)3NA a n d N - C B Z - A I a - p N P b o t h gave optimal hydrolysis at a b o u t p H 8.1-8.2. In considering the possibility t h a t more t h a n one enzyme m a y be attacking any one substrate, several inhibitors were tested using casein as substrate at

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pH Fig 1. Activity-pH curves for hydrolysis of substrates by Dover sole intestinal homogenates. (a) Casein F l - - r T , (b) Congo-red-elastin x - - - x and elastin-orcein C) O, (c) N-CBZ-AIa-pNP • . . . . . • and Suc(Ala)3 • 0,

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Table 1. Inhibition of Dover sole intestinal proteases Inhibitor Relative activity (?{,) concentration Inhibitor (ram) pH 6.9 pH 8.2 pH 10.0 100 100 100 PMSF 0.10 6 8 65 TPCK 0.17 77 80 84 TLCK 0.10 62 59 88 EDTA 9.40 78 63 57 N-bromosuccinimide 0.79 0 0 0 NEM 1.48 88 74 89 lodoacetate 5.03 95 94 89 PCM B 0.12 83 100 84

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different p H values. The results are s u m m a r i s e d in Table 1 a n d these indicated that there were only small differences in the extent of inhibition at the three pH values used except for P M S F which readily inhibited the more neutral proteases (at p H 6.9 a n d 8.2) but h a d a m u c h lesser effect on the protease activity at the alkaline p H (10.0). This effect is further studied at differing c o n c e n t r a t i o n s of P M S F (Fig. 3). On Sephadex G200 (Fig. 4) a m a j o r peak was produced with a n elution volume c o r r e s p o n d i n g to a molecular weight o f 19,500. The c o l u m n was calibrated using bovine serum a l b u m i n (mol. wt 67,000), o v a l b u m i n (45,000) c h y m o t r y p s i n A (25,000) a n d ribonuclease (13,370). A small subsidiary peak with a molecular weight in excess of 67,000 was also detected. In c h r o m a t o f o c u s i n g , a procedure in which

the proteins are eluted at approximately their isoelectric points, the m a i n elastase activity was eluted at pH 5.7 with a subsidiary peak at pH 5.3. In b o t h of those c h r o m a t o g r a p h i c procedures the casein hydrolysis procedure was used at pH 10.0 for assay of activity but the a p p r o p r i a t e fractions were also shown to be active towards elastin-orcein. It must also be noted that when elastase activity was measured using dyed substrates the highest activity was present in the lower regions of the gut in b o t h adult and juvenile fish. The diffuse nature of Dover sole pancreatic cells a n d the consequent secretion of enzymes over a wide area of gut mucosa together with the possible synthesis of elastase as a zymogen (as occurs in other species) may account for this result in as m u c h as the inactive zymogen requires time for exposure to activator proteases.

DISCUSSION In previous studies on Dover sole enzymes (Clark 1985) elastase was found to act within the alkaline region of proteolytic activity. High pro-

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Fig. 4. Gel filtration chromatography on Sephadex G200 of extracts of Dover sole intestine. Protease activity measured by hydrolysis of casein at pH 9.9 (O 0 ) and protein content of fractions (O . . . . . O). Elution o f standards: bovine serum albumin (B), ovalbumin (O), chymotrypsin (C) and ribonuclease (R).

Metabolism in marine flatfish--lit teolytic activity in the alkaline region has also been noted by other workers. For example Kamoi et al. (1975) detected high proteolytic activity at pH 10.5 in extracts from the pyloric caeca of young yellowtail (Seriola quinqueradiata). Assays for elastase activites in fish have been carried out using different substrates and at different pH values. Yoshinaka et al. (1982) reported optimal activity for a purified catfish enzyme to occur at pH 8.0 with succinyl-L-alanyl-L-alanylalanine p-nitroanilide as substrate, whereas Lansing et al. (1953) stated that pH 9.0 was optimal for the anglerfish enzyme using elastin as substrate. However, although pH 8.8 has been quoted as optimal for purified vertebrate elastases (Mandl, 1962; Shotton, 1970; Hartley and Shotton, 1971), optimum activities have been reported as high as pH 10.3 (Balo and Banga, 1950). It should also be noted that Lewis et al. (1956) found that the various optima recorded depended on the experimental buffer types. The present study was initially undertaken to investigate the high protease activity towards casein at pH 10.1 and, to this end, a number of different substrates were examined. It was shown that, although the buffer systems used in each case were the same at corresponding pH values, there was a wide difference in the optimal pH for activity, ranging from pH 8.1 to 9.8. The casein method (Fig. la) is very convenient as a general protease assay but it does not give any degree of specificity and hence the use of elastin-orcein and Congo-red-elastin (Fig. lb). Elastin-orcein gives a sharper profile than the Congored-elastin and this may indicate that the former substrate is more specific. This is in agreement with the observation of Mandl (1962) that orcein is stain specific for elastin and will not stain other tissue protein impurities in the substrate. The elastin substrates, dyed with either orcein or with Congo-red both have the disadvantage of being insoluble substrates and as such give relatively poor reproduceability because of differences in particle sizes and degree of dye substitution between different batches of the substrates. Also the reaction mixtures have to be rotated at a constant rate to ensure uniformity of reaction. It was therefore desirable to investigate artificial substrates and, in Fig. lc, the use of N-CBZ-Alap N P and Suc(Ala)3NA were found to give optimal values more than 1 pH unit different from the dyed elastins and, whilst substrates with different ionic configurations will have an effect on a pH-activity curve, this relatively large difference must raise the question as to whether the dyed elastins and the artificial substrates are measuring the same enzyme. From the shape of the curves in Fig. lc, although the N-CBZ-AIa-pNP give approximately the same optimal value, the peak is very much broader than that formed when Suc(Ala)3NA is used. This latter substrate would appear to have a good degree of specificity in as much as the pH-range in which it is hydrolysed is very narrow. Problems also exist with these artificial substrates; N-CBZ-Ala-p NP is unstable at alkaline pH values and both this substrate and Suc(AIa)3NA are not readily soluble in aqueous solution. To investigate the difference between dyed elastins which give pH optima at 9.4 and 9.8 and the artificial CB.P 81B/3 J

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substrates which give pH 8.1 and 8.2, the natural substrate elastin itself was examined. Fig. 2 shows the results of experiments in which the supernatant solution from the digests were assayed by either the Lowry or by the ninhydrin methods. With the Lowry assay, which measures soluble peptides containing phenolic amino acids released from the insoluble elastin substrate, a sharp optimum exists at pH 9.4, although, at about pH 8, a shoulder is also evident, presumably from other enzymes attacking the elastin. With the ninhydrin method of assay the optimum value is about pH8.3 and the curve is very flat between pH 6 and pH 9 indicating that exoproteases are probably acting on the elastin releasing free amino acids. This is substantiated by the fact that leucine aminopeptidase from Dover sole has an optimum pH value of 8.3 (Clark and Stark, in preparation). The effect of a range of inhibitors was also examined (Table 1). Using casein as substrate and assaying at different pH values, only PMSF showed any appreciable selectivity in degree of inhibition of hydrolysis of the substrate at different pH's. After pretreatment with PMSF there was a high degree of inhibition in the neutral regions but very much less at pH 10.0. This was examined further (Fig. 3) using slightly different pH-values and, by varying the inhibitor concentration, these conclusions were substantiated. Yoshinaka et al. (1983) reported that diisopropylfluorophosphate (DFP) and PMSF were potent inhibitors of elastase from catfish as measured using Suc(AIa)3NA at pH 8.0. It is interesting to note that when using orcein-elastin as substrate these authors used pH 8.8 for assays. These experiments on the catfish enzyme and the present inhibitor studies on the Dover sole enzymes are therefore not directly comparable. To obtain further information on the elastase activity in Dover sole intestine, some preliminary work was carried out by chromatographic methods. With molecular sieve chromatography using Sephadex G200 a major elastase activity was eluted at a volume corresponding to a molecular weight of 19,500 with a minor peak corresponding to a molecular weight of greater than 67,000 (Fig. 4). Cohen et al. (1981) have shown that a number of proteases from carp (Cyprinus carpio) including trypsin, chymotrypsin and elastase all have a molecular weight of 26,000 (Yoshinaka et al., 1983) and elastase B, the metalloprotein a molecular weight of 24,000 (Yoshinaka et al., 1984) as measured by polyacrylamide gel electrophoresis. In the present work on Dover sole elastase gel filtration chromatography indicated that the molecular weight was slightly lower than the above values. Chromatofocusing indicated that most of the Dover sole elastase had an isoelectric point in the region of pH 5.7, with some minor activity being eluted at pH 5.3. These experiments indicate that the assay of elastase activity in intestinal extracts may be measuring different enzyme activities and further comparative experiments will be required using highly purified enzymes to investigate the discrepancy between the pH of optimal activity of 9.4~9.8 for elastin and dyed elastins compared with the value of pH 8.1-8.2 for the artificial substrates.

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Acknowledgement--The authors wish to acknowledge the support given to this work by grants from the SERC (Marine Technology) to Dr J. Clark and Mr N. L. MacDonald.

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