Immunochemical determination of two trypsins in human duodenal juice

51

Clinica Chimica Acta, 94 (1979) 51-62 @ Elsevier/North-Holland Biomedical Press

CCA 10157

IMMUNOCHEMICAL DUODENAL JUICE

DETERMINATION

OF TWO TRYPSINS IN HUMAN

S. BORULF *, T. LINDBERG, B. BENEDIKTSSON and M. MANSSON Departments of Paediatrics and Experimental Hospital, 214 01 Malmii (Sweden)

Research,

University

of Lund, MalmZi General

(Received November lOth, 1978)

Summary

An application of electroimmunoassay to the separate determination of anionic and cationic trypsin in human duodenal juice is presented. The proportions of anionic to cationic immunoactive trypsin in duodenal juice from a group of children averaged 20 : 80. The ratio of immunoactive to esterolytitally (BAPNA) active trypsin averaged 1.6 : 1, indicating the presence of inactive forms of trypsin in duodenal juice. Introduction

A previous study [l] showed that, after electrophoretic separation in agarose gel of pancreatic enzymes in duodenal juice, at least two trypsins with different mobility at pH 8.6 can be identified: one anodal and one cathodal. The physiological interrelation of the two trypsins in duodenal juice is unknown; nor do we know whether defects with resulting decrease in enzymatic activity are confined to one or other of the two trypsin forms, or whether the defects are evenly distributed. Separate quantitative determination of the two trypsins in physiological and pathological conditions are therefore motivated. This study resulted in purification of the two trypsins from human duodenal juice and in the evolution of a method for immunochemical determination of the enzymes in intestinal contents. Materials Duodenal

juice

Duodenal juice was obtained from two healthy adults for the purification * To

whom correspondence

should be addressed.

52

and for elaboration of the immunochemical method. Juices for the application of the immunochemical method were obtained from a group of 9 children aged 8 months to 7 years studied for suspected malabsorption. Seven of the children had normal pancreatic exocrine function as tested by trypsin and amylase activity and also by the electrophoretic pattern of duodenal juice [ 11. Two had signs of pancreatic insufficiency with very low values for enzyme activity in the duodenal juice and absence of enzyme banding in agarose gel electrophoresis. The juice was collected into tubes in crushed ice, as described earlier [ 11, from the distal duodenum of patients in the fasting state and after an intake of water, 100-300 ml depending on age. It was kept frozen at -20°C until analyzed. Chemicals SP Sephadex C-50 and Sepharose 4-B, CNBr-activated, were obtained from Pharmacia, Uppsala, Sweden. Bovine albumin, cw-casein, n-benzoyl-DL-argininep-nitroanilide (BAPNA), n-benzoyl-DL-arginine-/3-naphthylamide (BANA), n-benzoyl-DL-phenylalanine /3-naphthyl ester (BEPNE), n-carbo-fl-naphthoxyL-phenylalanine (CNPA), n-benzoyltyrosine ethyl ester (BTEE) and soybean trypsin inhibitor (STI) were all purchased from Sigma Chemicals, St. Louis, U.S.A.; N-(3carboxypropionyl)-phenylalanine-p-nitroanilide (SUPHEPA) from Boehringer, Mannheim, F.R.G.; Blue starch polymer (BSP) e.g., Phadebas amylase test, from Pharmacia, Uppsala, Sweden; bovine trypsin (EC 3.4.4.4) from Fluka AG, Buchs, Switzerland; agarose from Marine Colloids Inc.-Miles Laboratories Ltd., Stoke Poges, U.K.; elastin powder from Worthington Biochemical Corp., Freehold, NY, U.S.A.; Trasylol bovine proteinase inhibitor from Bayer AC, Leverkusen, F.R.G.; di-isopropyl-fluorophosphate (DFP) from E. Merck, Darmstadt, F.R.G.; Freund’s complete adjuvant from DIFCO Lab., Detroit, U.S.A. All other chemicals used were of analytical grade. Methods Determination of enzyme activity. Trypsin esterolytic activity was measured with BAPNA as substrate [ 21, and bovine trypsin and the purified human trypsins as standards. One unit of activity was defined as an absorbance change of one A unit/min at 410 nm. Specific activity was calculated as units per mg protein. Proteolytic activity was determined with a single radial diffusion technique: 0.1% casein in 1.125% agarose and 0.2 M Tris-HCl, pH 7.9, containing 0.22 M CaCl,. After incubation (37”C, 6 h), the slide was immersed in 2% acetic acid, dried, and stained with Coomassie Brilliant Blue R 250. Chymotrypsin activity was determined with BTEE as substrate [3]. Carboxypeptidase activity was detected in gels by the method of Nagel et al. [4] with CNPA as substrate, adapted as a single radial diffusion technique in substrate-containing agarose gels. Elastase activity was similarly identified by the elastin digestion method [ 5,6]. Amylase activity was detected with BSP [ 71. In agarose gel electrophoresis, trypsin was identified by its BANA-splitting activity [l] and chymotrypsin was similarly identified with BEPNE and SUPHEPA [ 11.

53

Protein concentration was measured according to Lowry et al. [S] with bovine albumin as standard. PoiyacryZamide efectrophoresis was done at pH 4.5 according to Reisfeldt et al. [9] in 8% gels, omitting the urea. Four mA per tube was applied for 105 min. The gels were fixed in 10% trichloroacetic acid and the protein bands stained with Coomassie Brilliant Blue R 250 in 12.5% trichloroacetic acid. Agarose gel electrophoresis was done with 0.075 M sodium barbital buffer pH 8.6 containing 2 mmol/l calcium lactate ]lO]. Isolation of altodat and cathodal trypsin All procedures were done at 4°C unless otherwise stated. Step I. ethanol precipitation. The duodenal juice was incubated in ethanol (99.5%) l/10 v/v for 30 min at -ZO”C, centrifuged at 4”C, 9000 rpm. The supernatant was discarded and the precipitate dissolved in physiological saline. Step II. Ion-exchange chromatography on SP-Sephadex C-50. The protein solution from Step I was dialyzed for 2 h against 2 X 4 litres of starting buffer: 0.05 mmol/l sodium acetate 0.02 mol/l CaCl, (pH 4.3). It was applied to a column (2.5 X 22 cm) of SP-Sephadex C-50 previously equilibrated with starting buffer. The column was eluted with a linear NaCl concentration gradient obtained by connecting with the reservoir containing the eluting buffer a congruent vessel containing an equal amount of the same buffer with NaCl added to a molarity of 0.5. Step Ill Affinity chro~to~ap~y. A. Anionic trypsin, Soybean Sepharose 4-B. Soybean trypsin inhibitor (STI) was coupled to CNBr-activated Sepharose 4-B according to the manufacturer’s instructions. Ethanolamine, however, was replaced by Tris-HCl 0.1 mol/l (pH 8.0), 0.5 mol/l NaCl, and washing was done with this buffer alternating with 0.1 mol/l acetate buffer 0.5 mol/l NaCl (pH 4.0). The fractions from step II containing anionic trypsin were pooled and dialyzed for 2 h against 0.1 mol/l Tris-HCl, 0.05 mol/l CaCl*, 0.5 mol/l KCl (pH 7.5) and applied to a Soybean Sepharose 4-B column (2.0 X 16 cm) previously equilibrated with the same buffer. When unretarded proteins as read by Azsonrn had passed, the column was exposed to 0.1 molfl formic acid, 0.05 mol/l CaC12,0.5 mol/l KC1 (pH 2.5). The peak fractions were measured for BAPNA-splitting and proteolytic activity and kept at 4°C. B. Cationic trypsin., Trasybl Sepharose 4-B. Trasylol protease inhibitor (TPI) was coupled to CNBr-activated Sepharose 4-B, as described above. The fractions from Step II containing cationic trypsin were chromatographed at this material with a stepwise decrease of pH by the method of Johnson and Travis [ 111 as modified by Ohlsson and Skude [ 121. The resulting peak fractions were handled as described under A. Immunization procedure The t~psin-containing fractions from Step III were mixed with equal amounts of Freund’s complete adjuvant and injected intracutaneously and subscapularly into adult rabbits. Booster shots were given l-3 times every third week, after which the serum was harvested and IgG fractions isolated [13 J .

54

Assay procedures Immunoelectrophoresis according to Scheidegger [143 was run in 0.8% agarose in the ele~trophoresis buffer. Immunodiffusion anaiysis was performed by the double diffusion technique of Ouchterlony [ 151 in agarose gel and electrophoresis buffer. Crossed immunoelectrophoresis [16,17] and electroimmunoassay [18,19] were done in the electrophoresis buffer. ~eparution of standard for immunoc~emica~ assay Peak fractions from Step III were immediately inactivated with DFP [20], dialyzed against 1 mmol/l HCl for 2 X 3 h, and freeze-dried. The resulting powder was dissolved in 1 mmol/l HCl and stored at -20°C. Preparation of standard for enzymatic assay Peak fractions from Step III in various dilutions were immediately analysed for protein and for BAPNA-splitting activity. A standard curve was also obtained by mixing equal parts (v/v) of anionic and cationic trypsin. Results Isolation of human try~~ins Table I summarizes the procedures. Step II. Ion-exchange chromatography As Fig. 1 shows, two major peaks of trypsin activity are recovered. The trypsin content in the first peak to appear (A) gives one anodal band with BANAsplitting activity in agarose gel electro~hores~; the second (B) appears as two BANA-splitting bands on the cathodal side. In the Fig. 1 chromatogram, the localization of elastase, chymotrypsin, and carboxypeptidase is also shown. Step III. Affinity chromatography Fig. 2 gives the chromatograms. The results for peak A and peak B fractions TABLE I PURIFICATION

OF ANIONIC

(A) AND CATIONIC

(B) HUMAN TRYPSIN

Starting material: 264 ml duodenal juice.

step

Volume

Recovery

(nw

Total BAPNAsplitting activity (Units. U)

115

235.75

185.9

100

0.79

1

60 54

20.40 23.76

32.7 51.3

1s 26

1.60 2.16

2.0 2.7

11 15

1.1 1.95

24.2 49.5

13 27

(ml)

I. Ethanol precipitate

Total protein

(%)

Specific activity

Purlfication

(U/mg protein)

II. Sephadex C 50 column A. Anionic B. Cationic III. Affinity column A. Anionic (Soybean) B. Cationic (Trasylol)

22.0 25.3

24.2 32.2

55

NaCl(mol/l)

: g 0.3 ol G 0 : 0” (12 b In 2 0.1 10

20

30

40

50

60

70

60

ChT,

ti

* Fraction

90 c hTr

100

0

number

Fig. 1. Chromatography of ethanol-precipitated human duodenal juice on SP-Sephadex C-50. Fraction volume 5 ml. .-, absorbance at 280 nm: a-, BAPNA-splitting activity. ChTr, chymotrypsin; Cxp. carboxypeptidase A; El, elastase.

were similar: one single protein peak containing trypsin was eluted with the final buffers. These fractions were also analysed for elastase, carboxypeptidase, chymotrypsin, and amylase with negative results. Polyacrylamide gel electrophoresis showed one band for anionic and two for cationic trypsin (Fig. 3). Stability

of the enzymes

There was a marked difference in stability of the two trypsins in purified state. The anionic trypsin was remarkably unstable: at 4°C and pH 2.5, 90% of the BAPNA-splitting activity was lost after 6 h, and no enzyme band could be detected on electrophoresis (Coomassie Brilliant Blue). Freezing and thawing

Fraction

number

Fig. 2. Chromatography of peak A from SP-Sephadex C-50 on Soybean Sepharose 4-B (A) and of peak B from SP-Sephadex C-50 on Trasylol Sepharose 4-B (B). Fraction volume 5 ml. l -0, absorbance at 280 mn; *--a? BAPNA-splitting activity. ChTr. chymotrypsin.

56

Time

Fig. 3. Polyacryhmide disc electrophoresis (Soybean Sepharose). B. Cationic trypsin from anode (top) to cathode (bottom).

in 8% gels at pH 4.5. A. Anionic trypsin fraction from Step III fraction from Step III (Trasylol Sepharose). Running direction

Fig. 4. Esterolytically active and immunoactive trypsin in human duodenal juice incubated at 37OC. anionic trypsin: O- - - - - -0, *A, BAPNA-splitting activity (100% = 2.5 g/l); A- - - - - -a, immunosctive immunoactive cationic trypsin.

completely destroyed the enzyme. Raising the pH to 8.6 did not stabilize it. Inactivated anionic trypsin was more stable and retained its immunochemical reactivity for at least one month at -20°C. Repeated thawing destroyed the enzyme much more rapidly. On the other hand, the cationic trypsin was far more stable at low pH. In the purified state, it retained its enzymatic and immunochemical activity for at least 24 h at 4°C and also at -20°C (pH 2.5). Repeated thawing completely destroyed the enzyme. In inactivated form it is stable for months at -20°C. In the duodenal juice at -2O”C, both trypsins retained their activity for several months . At 37”C, the duodenal juice retained 60% of its initial 3APNA-splitting activity at 1 h, 25% at 2 h, and 10% at 4 h (Fig. 4). The anionic and cationic trypsin concentrations, determined by electroimmunoassay, decreased in a similar and parallel way (Fig. 4). Immunochemicai studies Rabbit antisera against the two trypsin preparations gave only one precipitation line with an indication of partial antigenic identity when tested against human duodenal juice in double diffusion in agarose gel. No precipitation occurred when bovine trypsin was tested against human anionic and cationic trypsin antisera. In immunoelectrophoresis, the antiserum against anionic trypsin gave two

t Fig. 5. Immunoelectrophoresis human anionic (A) and cationic

of human duodenal juice (healthy adult) with rabbit (B) trypsin run for 75 min at 20 V/cm at PH 8.6.

antibodies

against

precipitin lines when tested against duodenal juice: one strong in the anionic trypsin region and one corresponding to cationic trypsin. A similar cross-reaction, although weaker, was obtained for cationic trypsin antiserum tested in this way (Fig. 5). There were similar results when the purified enzymes were tested. Fig. 6 shows antigen-antibody crossed electrophoresis of human duodenal juice with rabbit antisera against the two human trypsins. Only one precipitate

A

Fig. 6. Crossed human anionic electrophoresis)

+

immunoelectrophoresis of duodenal juice (patient No. 9) with rabbit (A) (3%) and cationic (B) (2%) trypsin run for 70 min (electrophoresis) at 20 V/cm at pH 8.6.

antibodies against and 6 h (immuno-

58

is formed for each antiserum. In the cationic system, two peaks of equal height were seen, corresponding to the two cathodal trypsin bands in electrophoresis. Two peaks formed in the anionic system. The most anodal were always considerably smaller, and a corresponding trypsin band was not constantly found in electrophoresis. Immunochemical determination of trypsin Fig. 7 shows typical “rocket patterns” for anti-anionic and anti-cationic antibody-containing gels, tested against standard solution and against human duodenal juice from 9 children. Anodal trypsin formed one peak (Fig. 7a). At higher concentrations of cationic trypsin, a small anodal rocket emerged and fused with the cathodal one (Fig. 7b). The rockets formed in this system were distinct and easily measured.

,

STANDARD 123456789

123456789 STANDARD

b

Fig. 7. (a) Electroimmunoassay of 9 samples (10 ~1) of human duodenal iuiee with 2% rabbit antibodies against human anionic trypsin run for 6 h at 18 V/cm. Dilution of samples: Patient No. 1: l/Z: 2: l/l: 3 and 4: l/10; 5: l/5; 6: l/20; 7-9: l/10. Dilution of standard (0.62 g/l anionic DFP-trypsin): from left to right: l/4. l/8, l/16. l/3, l/6, l/12, l/24. b. Electroimmunoassay of 9 samples (10 ~1) of human duodenal juice with 2% rabbit antibodies against human cationic trypsin run for 6 h at 18 V/cm. Dilution of samples: Patient No. 1: l/2; 2: l/l; 3 and 4: l/10; 5: 115: 6-9: l/10. Dilution of standard (0.27 g/l cationic DFP-trvpsin): 1/10.1/6.1/5,1/2,1/1.

1.10 0.71

0.73

0.47

0.69

Unclassified malabsorption

Cow’s milk protein intolerance

UncIassified malabsorption

4m

Bm

8m

1~

1~

Or

?

8

9

* Standard curve: equaI parts (v/v) of anionic and cation& trypain. * * Protein determined by weight. *** Protein determined chemic&ly (9).

1.04

1.85

1.23

0.53

0.35

Sm

Unckusified malabsorption

0.87

Coeliaa disease

0.58

Hypoglycemia + subtotal pancreatectomy

0.87

OY

llm

5y

4

0.58

Unckssified malabsorption

0

0.03

2 ***

trypshl

OY XOm

8m

ly

3

0

Pancreatic insufficiency

0.02

1 **

Wl)

Bovine

1.04

0.71

1.10

1.84

0.52

0.85

0.86

0

a.03

* ***

tm

Human trypsin *

BAPNA-spIitting activity

6

7m

2y

2

Shwachman’s syndxome

Diagnosis

1.45 1.79 1.61

0.20 0.18 1.77

1.48

9.29

1.56 1.42

0.19 0.22

x.11 1.48

0.90

1.15

0.21 0.33

0.07

0.10

1.65

0.19

3.31

0.75

0.68

1.76

4.67

BAPNAdplitting activity

Immunoactivity

0.19

-

0.21

A/A + B

2.66

1.41

1.50

1.21 1.14

0

0.14

A*B (0)

0

0.11

B Cationic trYPain WI)

Ratio

0.65

0.27

0.29

Trace

0.03

A Anionic

Immunoactivity

ACTIVE AND IMMUNOACTIVE TRYPSIN IN HUMAN DUODENAL JUICE

6

Om

7~

Age

1

NO.

Patient

ESTEROLYTICALLY

TABLE II

60

Table II summarizes the results of trypsin determinations with immunochemical and enzymatic assays of the duodenal juices. The two patients with low trypsin activities had also low or zero values for immunoactive trypsins. The ratio between immunoactive and enzymatic assayable trypsin averaged 1.6 :1 in the patients who had no evidence of pancreatic insufficiency, whereas in patient No. 1 who had pancreatic insufficiency, the ratio was three times as high 4.7 : 1. The proportion of immunoactive anionic trypsin was throughout about 20%, except for patient No. 5 (who had coeliac disease), where it was 10%. Discussion The presence of two trypsins, one anionic and one cationic, in human exocrine pancreas has long been established [Zl--251. In attempts to isolate these enzymes from the pancreas or from pancreatic juice, the anionic form proved more labile than the cationic form [24,26], Our experience of purifications from duodenal juice sustains this. It is thus of the utmost importance for the anionic trypsin to be taken care of immediately, as a major part of it is destroyed within 6 h at 4°C and at pH 2.5-8.6. Moreover, freezing and thawing destroys the isolated enzyme completely. Autolysis of the enzyme molecule, as suggested by Mallory and Travis [ 241, seems to be the most probable explanation of this phenomenon. In our (and others [26,27]) experience, the trypsins are more stable in the duodenal juice at low temperature, e.g. for several months at -20°C. Colomb et al. [28] reported that at 37°C about 50% of the trypsin activity (esterolyti~) is present in duodenal juice after 1 h and that the addition of 20 mmol/l calcium to the duodenal juice will stabilize the enzymes. They ascribed this inactivation to autolysis mainly of the anionic trypsin. We found a similar loss of the esterolytic activity in duodenal juice at 37°C. At the same time, the immunoactive anionic and cationic trypsins decreased proportionally to an equal degree. Thus it seems that the inactivation at 37°C is due to autolysis of both trypsins. Mallory and Travis [29] reported equal inhibition of casein proteolysis for anionic and cationic trypsin by soybean trypsin inhibitor and Kunitz bovine trypsin inhibitor. The successful use of soybean trypsin inhibitor as an affinity ligand for anionic trypsin despite the presence of chymotrypsin is perhaps surprising. Possibly, the explanation is that the chymot~psin is of type II and thus can be presumed to have a weaker affinity for soybean trypsin inhibitor than has type I. The quantitative relationship between the two trypsins in their enzymatic activities has been studied, but so far only in extracts from pancreas and in pancreatic juice [ 24,26,28,29]. Mallory and Travis [ 291 reported the anionic trypsin to comprise 3-4% of total proteolytic capacity in pancreatic extracts compared with 31% for cationic trypsin. They also suggest that the anionic trypsin is present in much lower concentrations than the cationic trypsin [ 241. Figarella [ 261 reported that their trypsin, corresponding to cationic trypsin, represents the major part (65%) of potential trypsin (tosylarginine methyl ester-

61

splitting) activity of the pancreatic juice. We found that in duodenal juice, the immunoactivity ratio of anionic to cationic trypsin was 20 : 80. After Sephadex fractionation, we found the BAPNA-splitting activity ratio of anionic to cationic trypsin to be 39 : 61. In the purified state, the ratio in BAPNA-splitting activity per g protein of anionic to cationic trypsin was 46 : 54. In proteolytic (casein hydrolyzing) activity the ratio was about the same 44 : 56. As the anionic trypsin seems to be as stable as the cationic in duodenal juice, and as a considerable part of the total proteolytic and also esterolytic capacity of the duodenal juice can be ascribed to this enzyme, it is probable that both enzymes are of significant importance in protein digestion, a matter that has been questioned [ 291. Our immunochemical studies show a weak cross-reaction between anionic trypsin and anticationic trypsin antibodies, as previously demonstrated [ 24,251. We could show a cross-reaction also between cationic trypsin and antianionic trypsin antibodies. In crossed immunoelectrophoresis of duodenal juice vs. anticationic trypsin antibodies, two almost equally large precipitin peaks are formed. These peaks correspond to the two cathodal bands constantly recognized in electrophoresis of duodenal juice [l] and also of purified fractions of cationic trypsin. Both protein bands show distinct BANA and BAPNA-splitting activity, which indicates that both peaks contain active trypsin. For anionic trypsin, the crossed immunoelectrophoresis reveals an additional peak on the anodal slope of the precipitate with a fusion indicating immunoidentity. The nature of this additional peak is not clear, but it possibly represents an active enzyme, as we have found an additional anodal BANA- and BAPNA-splitting band upon electrophoresis in 45% of individuals [ 11. The present study shows the usefulness of electroimmunoassay (EIA) for separate quantitation of anionic and cationic trypsin in duodenal juice. Parallel analyses of the duodenal juice with electroimmunoassay (EIA) and enzymatic assays might clarify whether anionic and cationic trypsin are present in inactive forms. Earlier attempts to quantify trypsin immunochemically in duodenal juice have been made with radial immunodiffusion techniques and included the cationic trypsin only [30]. These authors found a ratio of about 1.2 : 1 between immunoactivity and esterolytic activity for cationic trypsin. Our corresponding figure is 1.3 : 1. If anionic trypsin is included, the figure increases to 1.6 : 1. In our patients, substantial discrepancies in the same direction, but of different magnitude, were established between immunochemical and enzymatic estimates. This suggests the presence of inactive forms of the enzymes in duodenal juice. It is noteworthy that, in patient No. 1, whose pancreatic function has not ceased completely, the proportion of immunoactive enzyme is greater. Moreover, the ratio between anionic and cationic trypsin is not constant, as is demonstrated in patient No. 5 with coeliac disease. Further studies in many more patients are necessary to decide the importance of these discrepancies in relation to normal and pathological conditions.

62

Acknowledgement We are very much indebted to Dr. K. Ohlsson for valuable methodological advice. Excellent technical assistance was given by Mrs. L. Hansson. This work was supported by The Swedish Medical Research Council (Project No. 5143 and 5364), The Swedish Baby Food Industry Fund for Nutritional Research, The Swedish Nutrition Foundation, Semper Nutrition Foundation, and the Albert Plhlsson Fund. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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