The effect of acclimation temperature, assay temperature, and ration on the specific activity of trypsin and chymotrypsin from rainbow trout (Salmo gairdneri)

The effect of acclimation temperature, assay temperature, and ration on the specific activity of trypsin and chymotrypsin from rainbow trout (Salmo gairdneri)

Comp. Biochern. Physiol. Vol. 73B, No. 3, pp. 631 to 634, 1982 0305-0491/82/110631-04103.00/0 Pergamon Press Ltd Printed in Great Britain. THE EFFE...

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Comp. Biochern. Physiol. Vol. 73B, No. 3, pp. 631 to 634, 1982

0305-0491/82/110631-04103.00/0 Pergamon Press Ltd

Printed in Great Britain.


(SALMO GAIRDNERI) J. M. MCLEESE and E. DON STEVENS Department of Zoology, University of Guelph, Guelph, Ontario, Canada N I G 2Wl (Received 11 March 1982)

Abstract--l. Specific activity of trypsin and chymotrypsin from rainbow trout acclimated to 10 or 15°C and fed ad lib or low ration was higher from anterior caeca than from posterior caeca. 2. Ration had no effect on the specific activity of either enzyme even though those fed ad lib consumed more than twice as much as those on the low ration diet. 3. Acclimation temperature had no significant effect on the specific activity of either enzyme, thus we did not observe thermal compensation. 4. The activation energy and Ql0 were 11,000 and 2.0 for trypsin and were 9000 and 1.78 for chymotrypsin. Fish were sampled in three groups, at weekly intervals. For the first sampling, the two 15°C tanks were starved for 48 hr, and the 10°C tank starved for 72 hr. For the following two groups, all tanks were starved for 72 hr, to insure empty guts. Fish were killed by cervical dislocation, were weighed, and the viscera removed from esophagus to anus. Extraneous fat (except that immediately around individual caeca), the liver and gall bladder were quickly removed and discarded. The intestine bearing the pyloric caeca was freed, and divided into two sections. The anterior portion was that on which the caeca extended from both the ventral and dorsal aspects of the intestine, while the posterior portion was that on which the caeca extended only from the dorsal aspect. The posterior portion was two to three times longer than the anterior portion. Each section was frozen by freeze clamping in liquid nitrogen, then wrapped in aluminum foil for storage. Samples were stored at -40°C. For analysis, samples were removed from the freezer and thawed overnight at 4°C. Samples were weighed, and nine volumes of Tris buffer (Sigma TRIZMA, HCI and TRIZMA base), pH 8.0, containing 0.04 M calcium chloride, was added. This mixture was homogenized by Polytron, and the homogenate was centrifuged at 0°C for 20 min at 13,500.q. The supernatant was removed and further diluted with 7 vol. of the same Tris buffer for trypsin specific activity, and with 3 vol. for chymotrypsin specific activity. Specific activity of trypsin was assayed using the synthetic substrate :t-benzoyI-DL-arginine-p-nitroanilide.HCl (BAPNA/, according to the method of Preiser et aL (19751. Buffer, rather than acid albumin was used since fish protease is acid denaturable (Croston, 1960). All quantities were doubled to give sufficient volume. Chymotrypsin specific activity was assayed by the method of Erlanger et al. (1966), using glutaryl-L-phenylalanine-p-nitroanilide (GPNA). Incubation was for 10 min at 5, 10, and 15°C. The coloured product was read at 550 nm (trypsin) or 410 nm (chymotrypsin) on a Zeiss PMQ-II spectrophotometer. The absorbances were compared to those given by standard curves of p-nitroanilide to determine the concentration of product released. The remainder of the diluted sample was frozen at - 4 0 ° C following these determinations and stored for protein analysis. Protein was estimated by the method of Lowry et al.


T e m p e r a t u r e markedly alters the rate of fish growth, so that for rainbow trout, with each increase in holding temperature of 4°C between 7 and 15°C, there is a doubling of growth (Slinger & Cho, 1978). Thus, fish held at a higher temperature must process more food and one might predict that there would be some modifications of the digestive enzymes involved in dealing with the increase in the a m o u n t of food that must be processed, O n the other hand, there are n u m e r o u s examples given by H o c h a c h k a and his colleagues to show that fish held at lower temperatures alter enzymes or their modulation in such a m a n n e r so as to compensate for the direct effects of temperature on reaction rate. Thermal compensation of digestive process in fish has not been well studied. It has been shown that the specific activity of pepsin is thermally compensated in brook trout (Owen & Wiggs, 1971), as is gastric secretion in brown bullheads (Smit, 1967). Hofer et al. (1975) reported that the Michaelis constant (i.e. the Kin) of trypsin of several fish species is nearly independent of assay temperature. In the present study we examined the extent of thermal compensation of trypsin and chymotrypsin, two i m p o r t a n t intestinal proteases, in rainbow trout. MATERIALS AND METHODS

Rainbow trout (Salmo gairdneri Richardson) were placed into three tanks for temperature acclimation. Twenty-four fish were acclimated to 10°C and fed ad lib (tank-10ad). Twenty-three fish were acclimated to 15°C and fed ad lib (tank-15ad), while 26 fish were acclimated to 15°C and fed the same weight of feed as those acclimated to 10°C (tank-15pr). All fish were fed diet No. 5 of Cho et al. (1974) three times daily at 9a.m, 12p.m., and 4p.m. for the 3 week acclimation period. The feed was a practical diet containing 18% fat, 40~o protein and 5% carbohydrate, the remainder being ash and fiber. 631


J.M. M('LIEs[3 and E. DoN STEVENS Table I. Ration as a percent of body weight at the start of the trial and growth for lish held at the two temperatures and the two ration levels. Body weights at the end of the trial are given as mean + SEM Accl temp


n 10-ad 15-ad 15-pr

10 15 15

Start of trial 137.50 11)8.7 146.92

24 23 26

end of trial 154.81 _+ 11.19 146.35 + 10.36 148.23 + 3.27

REStJ LTS (A). Growth rate T r o u t held at 15'C ate m o r e than twice as m u c h food a n d gained a l m o s t three times as m u c h weight as t h o s e held at 10°C w h e n b o t h g r o u p s were fed ad libitum (Table 1). T r o u t held at 15°C but fed a ration similar in a m o u n t to the m a x i m u m a m o u n t that those held at 10°C can eat just m a i n t a i n e d b o d y weight w h e r e a s those held at lff'C gained weight (Table lt.








ASSAY T E M P 1 0 ° C

80 A ¢ B

0.89 1.79 0.62

In o r d e r to eliminate the possibility that the effect of acclimation t e m p e r a t u r e ~ a s due to different a m o u n t s fed at different temperatures, the fish held at 1 5 C were fed at two ration levels: ad lib a n d at a l o ~ level {approximately the same as that fed to the tish held at 10C). Even t h o u g h the ration level at the ad lib level was almost twice as much as that at the lower

T h e caeca lie in rows along the intestine and were divided into two segments: a n t e r i o r (those closest to .

(I!i, increase) 12.6 34.6 0.89

(el. The
(B). D!ff~,rences between anterior and posterior caeca


Ration da~

the s t o m a c h ) a n d posterior. T h e specific activity of trypsin from a n t e r i o r s e g m e n t s was s i g n i f i c a n t b g r e a t e r than that from the posterxor s e g m e n t s for all treatments, and at all assay t e m p e r a t u r e s {Fig. 1). This t r e n d was identical for c h y m o t r y p s i n , but not all differences were significant (Fig. 1). T h e r e were no significant differences between a n t e r i o r a n d posterior s e g m e n t s from tank-10ad or from tank-15pr when assayed at 5"C, or from tank-15ad when assayed at 15=( '. T h u s the caeca in the anterior segment of the gut have greater specific activity of both trypsin and chym o t r y p s i n and this effect is i n d e p e n d e n t of the acclim a t i o n t e m p e r a t u r e or the ration level. In most subsequent c o m p a r i s o n s the data from the a n t e r i o r segment are used to e x a m i n e the effect of assay t e m p e r a lure, a c c l i m a t i o n t e m p e r a t u r e , and ration. In all cases the results are similar for the posterior segment.

(1951 ) with following modifications: the standard curve of bovine serum albumin was diluted with Tris buffer, pH 8.0, containing 0.0395 M calcium chloride so that the concentration of calcium chloride was equivalent to the sample concentration. Following the addition of the Folin's reagent, each tube was centrifuged for 10 rain in a table top clinical centrifuge to remove precipitate. The resultant supernatant was the read at 550 nm. All chemicals for the enzyme assays, and the serum albumin, where purchased from Sigma, while the rest for the protein determination were obtained from Fisher. Statistical analyses were by Student's t-test (Steel & Torrie. 1960).

tOO .

Weight gain

Average body weight (g)




I!lI j A P


























ASSAY T E M P 10°C ASSAY TEMP• 5 ° C 2.0












Fig. 1. The specific activity of trypsin (top panels) and chymotrypsin {bottom panels) from rainbow trom anterior caeca (A) compared to posterior caeca (P) at the three assay temperatures. Labels below each pair indicate the acclimation temperature and ration level (ad = ad lib). Numbers above each label indicate a probability of the difference between anterior and posterior caeca being dne to chance.

Specific activity of trout trypsin and chymotrypsin


Table 2. The effect of ration level on specific activities of trypsin and chymotrypsin from pyloric caeca of rainbow trout held at 15°C. Enzyme activity was assayed at three temperatures (5, 10 and 15°C) and the ceaca were divided into two segments (anterior and posterior)

Treatment Ration (g/d/fish) Trypsin specific activity anterior caeca 5C 10 15 posterior caeca 5 10 15 Chymotrypsin specific activity anterior caeca 5C 10 15 posterior caeca 5 10 15

Tank 15-ad

Tank 15-pr

Difference as ~o of tank 15-ad

ad lib 1.951

low ration 0.9059


45.75 68.67 94.06 38.51 55.92 75.58

41.09 61.78 85.43 34.58 49.62 69.18

10.2 10 9.2 10.2 11.3 8.5

1.787 2.428 3.364 1.578 2.024 2.885

ration level (Table 1), the specific activities were the same (Table 2). Specific activities of trypsin from fish at the low ration level were about 10~o less than those at the ad lib level. Specific activities of chymotrypsin from fish at the low ration level ranges from 1Vo more to 11~,; less than those at the ad lib level. However none of the above changes were significantly different. Thus the results from the two ration levels are combined to test the effects of acclimation and assay temperature. (D). The effect of acclimation temperature The effect of acclimation temperature was estimated by comparing the results from fish held at 10°C

1.768 2.246 2.987 1.573 2.072 2.760

1.1 7.5 11.2 0.32 - 2.4 4.3

to the combined results from the fish held at 15~'C. The specific activity of trypsin was the same at the two acclimation temperatures (Fig. 2) for caeca from both the anterior and posterior segments of the intestine and at all three assay temperatures. The specific activity of chymotrypsin was the same at the two acclimation temperatures for caeca from the anterior segment of the intestine at all three assay temperatures, and for caeca from the posterior segment of the intestine when assayed at 5°C. However, chymotrypsin specific activity was significantly greater at the higher acclimation temperature in the posterior caeca when assayed at either 10°C (P < 0.03) or 15cC (P < 0.01). (E). The effect of assay temperature







2 o






2 1 0

Fig. 2. The effect of acclimation temperature (10 and 15°C) on the specific activity of trypsin and chymotrypsin from trout caeca. Asterisks indicate a significant difference related to acclimation temperature.

Both trypsin and chymotrypsin specific activity increased with an increase in assay temperature. The data are presented as Arrhenius plots in Fig. 3. The apparent activation energy was about l l,000cal for trypsin and about 9000 cal for chymotrypsin. The Q~0 from 5 to 15°C for trypsin was 2.0 and was slightly higher at lower temperatures (2.1 from 5 to 10c'C, and 1.9 from 10 to 15°C). The Q~o from 5 to 15°C for chymotrypsin was 1.78 and was slightly lower at lower temperatures (1.69 from 5 to 10°C, and 1.88 from 10 to 15°C). DISCUSSION

The pancreatic tissue of the rainbow trout is located in the fat and mesentery surrounding the pyloric caeca (Weinreb & Bilstad, 1955). Our results suggest that it is relatively evenly spread among the anterior and posterior portions of the caeca. However, as might be expected from a comparison with species possessing a distinct pancreas, there is more enzyme, and hence probably more pancreatic tissue in the anterior section. Hofer (1979) showed that an increase in animal components over detrital components in the diet of


J.M. M('Lt!ESE and E. DON STEVENS ! ~



\ _~








Ackm)wledgements This study is supported b) an NSERC operating grant to EDS. JMM is supported by an NSERC graduate scholarship. We thank Drs J. W. Hilton and L. Lowe-Jinde for comments on the manuscript and especially thank Dr Hilton for providing space, animals, and holding facilities.

2.4 o ,'7

plots are within the range of activation energies found for proteases by other investigators (Owen & Wiggs, 1971: Fabian, M o l n a r & Tolg, 1963). The Q~o values are close to two, within the the range expected for a non-compensated enzymatic reaction. Hofer et ~d. (1975) reported that many fish species (including rainbow trout) exhibit a relatively temperature independent K,, suggesting that compensation may occur at this level. We are examining kinetic properties ol these enzymes in order to get at this aspect of the question.



"~o 0.00350




Fig. 3. Arrhenius plots of the specific activity of trypsin and chymotrypsin from trout caeca.

roach (Rutilus rutilus) and rudd (Scardinius erythrophthahnus) resulted in an increase in protease activity in the intestinal contents. Using rinsed homogenized intestine, Kawai & Ikeda (1972) showed that in carp (Cyprinus carpio) protease specific activity increases when the level of protein is increased. These results are in contrast to those of the present study in which only a small and insignificant increase was obtained. Ration level therefore was not an influencing factor in the response of these enzymes to acclimation temperature. C o m p e n s a t i o n to temperature in fish digestive processes has been observed. Owen & Wiggs (19711 showed that pepsin activity was 30°,~, greater in cold acclimated b r o o k trout (Sah, elinus Jbntinalis) compared to that of warm acclimated fish when assayed at the same temperature. Even though b o t h trypsin and chymotrypsin are also i m p o r t a n t to protein digestion we did not observe a similar increase in activity in rainbow trout. No differences were observed for trypsin for fish acclimated to l0 and 15°C when assayed at any temperature. In a few cases we observed some evidence of slight inverse compensation for chymotrypsin (i.e. slightly lower in cold acclimated fish). Thus the specific activities of trypsin and chymotrypsin are not increased in cold acclimated rainbow trout. Smit (1967) showed that the rate of gastric secretion in brown bullheads (lctalurus nebulosus) is compensated for temperature. The logistic difficulty in measuring the secretion rates in fish with a diffuse pancreas precludes attempting this experiment in rainbow trout to look at trypsin and chymotrypsin secretion. Activation energies determined from Arrhenius

REFERENCES CHO C. Y., BAYLEY H. S. & SLINGt-R S. J. (19741 Partial replacement of herring meal with soybean meal and other changes in a diet for rainbow trout (Salmo gtairdneri). J. Fish. Res. Bd. Cwz, 31, 1523-1528. CROSTON C. B. (1960) Tryptic enzymes of Chinook salmon. Archs Biochem. Biophys. 89, 202 206 ERLANGER B. F., EDEL F. & C(X)PER A. G. (1966} The action of chymotrypsin on two new chromogenic substrates. Archs Biochem. Biophys. 115, 206 210. FABIAN GY., MOLNAR GY. & TOL(; l. (1963) Comparative data and enzyme kinetic calculations on changes caused by temperature in the duration of gastric digestion of some predatory fishes. Acta Biol. Hung. 14, 123 129. HOFER R. (1979) The adaptation of digestive enzymes to temperature, season and diet in roach. Rutilus rutilus and rudd Scardinius erythrophthahmlus: proteases. J. Fish Biol. 15, 373 379. HOFER R. M., LADURNtR M., GA'ITRINGERA. & WEISkR W'. (1975) Relationship between the temperature preferenda of fishes, amphibians and reptiles, and the substrate affinities of their trypsins. J. comp. Physiol. 99. 345 355. KAWAI S.-I. & IKEDA S. (1972) Studies on digestive enzymes of fishes II. Effect of dietary change on the activities of digestive enzymes in carp intestine. Bull. Jap. Soc. Sci. Fish. 38, 265 270, LOWR', O. H., ROSkBROU(;It N. J., FARR A. L. & RANI)ALI R. J. (19511 Protein measurement with Folin phenol reagent. J. biol. Che,1. 193, 265 275. OWEN T. G. & WIc;(;s A..I. 11971) Thermal compensation in the stomach of the brook trout {Sah'elitms .[bntinali.~ Mitchill). Conlp. Biochem. Physiol. 40B, 465 473. PREISER H., S('I'tMITZ J.. MAESTRA(('I D. & CRANF R. K (1975) Modification of an assay for tr)psin and its application for the estimation of enteropeptidase. Clin. Chim. Acta59, 169 175. SL1NGI-:R S. J. & CHO C. ~. (1978} Fish thrming Research needs and prospects, tti#hli#hts q/ 4gricultural Research in Ontario. Vol. 1, pp. 13 16. SMIT M. (1967) Influence of temperature on the rate of gastric juice secretion in the brown bullhead, lctalurus nehulosus. Comp. Biochem. Ph~'siol. 21, 125 132. STEI':L R. G. D. & TORRll~. J. H. (1960) Principles and Procedures qf Stutistics. McGraw-Hill, Toronto. WHNREB E. L. & BILSrAD N. M. (1955) Histology of the digestive tract and adjacent structures of the rainbow trout, Salmo ,qairdneri irideus. Copeia 194 204,