ANALYTICAL
98, 438-444
BIOCHLMISTRY
Two Simple
JOHN
A.
(1979)
Quantitative Assays for Trypsin Using Immobilized Trypsin GATEHOUSE
AND
ANGHARAD
Received
March
M. R.
Inhibitors
GATEHOUSE
7, 1979
Two new methods for quantitative assay of trypsin inhibitors, suitable for large numbers ofsamples, are described. The assay methods use trypsin-Sepharose conjugates incorporated into agarose gel slabs. Trypsin inhibitors are allowed to diffuse into, or are electrophoretically moved through, the slabs, and the consequent areas of inactivation of immobilized trypsin are visualized using a histochemical enzyme substrate. Quantitation of the trypsin inhibitor content of samples can be made on the basis of the inactivated areas. The limit of detection is 1-2 pg of soybean trypsin inhibitor and determinations are reproducible to 10% or better. Measured trypsin inhibitor contents of several legume species and varieties agree with spectrophotometric determinations.
Protein trypsin inhibitors are well known as potential antimetabolic components of many plant tissues, and as such their presence may be undesirable: for example, raw soybeans are antinutritional to animals due to, among other things, the high concentration of trypsin inhibitors they contain (1.2). On the other hand, in the native plant trypsin inhibitors may confer desirable properties; it has been hypothesized that trypsin inhibitors (as the major proteinase inhibitors) are responsible for resistance of plant material toward attack by insects (3-5) fungi, and bacteria (6,7). Recently it has been shown that an elevated level of trypsin inhibitors was responsible in the laboratory for the resistance of the seeds of a variety of cowpea (Vigrza ung:lriculatu) toward the bruchid beetle Cal1osobtwc~hu.s rnaculatuLs (8). Further, the antimetabolic properties of trypsin inhibitors may be removed by cooking or processing (9,10), and since the proteins as a rule have a high sulfur content (11) they may make a significant contribution to the nutritional value of legumes, which are deficient in sulfur amino acids (12,13), as animal or human foodstuffs. 0003.2697/79/140438-07$02.00/O Copyright All rights
I 1979 by Academc Press. Lnc. 01. reproductmn ,n any form reserved.
438
Irrespective of whether trypsin inhibitors are considered as desirable or undesirable components of plant materials, the levels at which they are present need to be estimated for quality control, or for comparisons between species and varieties. It is thus useful to have a quantitative assay for trypsin inhibitory content which can be applied to the large numbers of samples involved in screening programs. While a number of reasonably accurate methods are available for quantitative estimation of trypsin inhibitors, mainly by measuring the reduction in the rate of hydrolysis by trypsin of natural or artificial substrates caused by the inhibitor (14-16), they are time consuming. On the other hand, several rapid methods exist for the detection of trypsin inhibitors (17- 19) but none is quantitative. The present paper describes two simple, quick, quantitative methods for estimation of trypsin inhibitors based on diffusion or electrophoresis of the inhibitors into gels containing immobilized trypsin. The methods may be applied to small samples (10 mg of plant material or less) and are suitable for estimating the inhibitor content
QUANTITATION
OF TRYPSIN
of single seeds, or single cotyledons, with sufficient accuracy for plant breeding purposes. MATERIALS
AND METHODS
Muterids. Trypsin (Type I, from bovine pancreas), soybean trypsin inhibitor (Type l-S), and the enzyme substrates cu-Nbenzoyl - DL - arginine -p - nitroanilide-HCl (BAPNA)’ and a-N-benzoyl-DL-arginineP-naphthylamide- HCl (BAN A) were obtained from Sigma (London) Chemical Company Ltd., Poole, Dorset, England. Fast blue B salt (diazonium salt of o-dianisidine) and cyanogen bromide (pure) were obtained from Koch-Light Laboratories Ltd., Colnbrook, Buckinghamshire, England. Agarose, immunodiffusion plates, and well punches were purchased from Miles Laboratories Ltd., Slough, England, and the Sepharose 4B was supplied by Pharmacia Fine Chemicals, Uppsala, Sweden. Buffer components and reagents were obtained from British Drug Houses (BDH) Ltd., Poole, Dorset, England, and were of analytical grade when necessary. Merhods. Trypsin was immobilized by coupling onto cyanogen bromide-activated Sepharose 4B (approximately 2.5 mg protein/ml of gel). Both the activation and subsequent coupling were carried out according to the method of March et ~11.(20). Prior to use the coupled trypsin was diluted with distilled water (1:2) to form a slurry of known enzyme concentration. The assay gels were prepared by dissolving agarose in 1 vol of boiling water on a thermostatically controlled magnetic stirrer and then adding 1 vol of buffer to give a final concentration of 1% agarose in 0.05 M Tris-HCl, 0.02 M CaCl,, pH 8.2. The gel mixture was equilibrated at 55°C and the trypsin/Sepharose slurry (equilibrated at 55°C for 10 min) was added and mixed ’ Abbreviations used: arginine-p-nitroanilide-HCI; arginine-P-naphthylamide-HCI.
BAPNA. BANA,
a-N-benzoyl-ol.a-A’-benzoyl-oL-
439
INHIBITORS
thoroughly (25 ~1 of slurry per milliliter of 1% agarose was found to be optimum for the levels of trypsin inhibitor assayed). Gels were cast immediately. Diffusion gels of loml volume were cast in Miles immunodiffusion plates. Slab gels for electrophoresis were of 30-ml volume and were cast on oven-dried glass plates (110 x 205 mm) precoated with 0.5% agarose in 50% aqueous methanol. Trypsin inhibitor samples were routinely prepared by extracting 20-mg samples of meal in 500 ~1 of 0.05 M TrisHCl, 0.02 M CaCl, buffer, pH 8.2, for 4 h at room temperature. The extracts were centrifuged for 5 min in a bench centrifuge (9OOOg)and lo- or 20+1 samples of the supernatants were applied to wells punched through the gel using a 3- or 4-mmdiameter punch. Either cowpea trypsin inhibitor, purified by affinity chromatography (21), or soybean trypsin inhibitor standards were run on each gel. Diffusion plates were left 16 h at room temperature and then transferred to precoated agarose glassplates. Rocket electrophoresis was carried out on a flatbed electrophoresis apparatus with a water-cooled platen, using barbitone-sodium buffer, pH 8.6 (22). The gel was allowed to run overnight at 5 V/cm. Following electrophoresis or diffusion the gels were rinsed in distilled water and subsequently pressed and air-dried (23). They were then incubated with the histochemical substrate and stain (30 mg fast blue B salt dissolved in 30 ml sodium acetate (0.1 M, pH 6.5), 24 ml sodium chloride (0.14 M), and 3 ml potassium cyanide (0.02 M), to which was added 24 mg BANA dissolved in 3 ml methanol (24); the reagent must be used immediately). Maximum staining was achieved in approximately 30 min after which time the gels were again rinsed and finally pressed and airdried. RESULTS Sepharose-coupled trypsin was shown to be reasonably stable under the conditions used for incorporation into agarose gels by
440
GATEHOUSE
AND
GATEHOUSE
FIG. 1. Diffusion assays for trypsin inhibitors. Wells standards, 0, 2, 4. 6, 8. and 10 fig inhibitor, respectively. 10 Pg.
comparing the rates of BAPNA hydrolysis by the conjugate on incubation at 55°C and at room temperature; no significant difference was observed after 15 min. Further, trypsin activity could be detected in an agarose gel slab incorporating the Sepharose-trypsin conjugate by incubation in a solution of BAPNA, on which the solution rapidly turned yellow due to release of pnitroaniline. However, to locate the active trypsin a histochemical substrate, BANA, that gave an insoluble colored product on trypsin hydrolysis had to be employed. An agarose gel slab containing trypsin-sepharose was pressed, dried, and incubated with BANA-fast blue B stain solution. Trypsin activity could now be seen to be located at points, giving the agarose gel a speckled appearance like a poorly printed photograph. The red color produced by hydrolysis of BANA is fixed in the gel and will not wash out; the gel may thus be washed. pressed. and dried without altering the stained areas. The initial method devised for assay of trypsin inhibitors was by radial diffusion. If a solution of trypsin inhibitor is placed in a circular well punched in the agaroseSepharose- trypsin gel, the inhibitor will diffuse outward in all directions. As it encounters trypsin molecules, the inhibitor will bind to them and inactivate them. Since the trypsin-inhibitor binding is strong and
1. 2, 3. 4. 5, and 6: cowpea trypsin Wells 7 and 8: soybean trypsin
inhibitor inhibitor.
kinetically stable at the pH used (11) inactivation is effectively permanent and the inhibitor cannot diffuse away once bound. The unbound inhibitor will continue diffusing until all of it has complexed with trypsin, the result being a circular area around the well where the immobilized trypsin has bound inhibitor and been in1. 5
FIG. 2. Calibration of diffusion trypsin inhibitor (CPTI) and hibitor (SBTI) standards.
assays, using cowpea soybean trypsin in-
QUANTITATIiON
OF TRYPSIN
activated. If the agarose gel is now incubated with the histochemical trypsin substrate BANA the effect of the inactivation is seen as a clear area round the well where no trypsin-catalyzed hydrolysis has occurred (Fig. 1). The area of the circle is proportional to the amount of inhibitor originally placed in the well; the diameter of the circles may be measured with dividers and the areas calculated. Typical clalibrations using soybean trypsin inhibitor and cowpea trypsin inhibitor are given in Fig. 2. The unknown trypsin inhibitor content of samples may now be estimated from the standard curve. The reproducibility of this assay was tested using purified cowpea and soybean trypsin inhibitors: on the same
batch of slabs reproducibility is good and values may be estimated with an error of less than 10%. The standard curve, however, was found to vary slightly from one batch of experiments to another, due to nonreproducibility in making up the slurry of trypsin--Sepharose. The method is of general applicability, as shown by the estimations of inhibitor content of seeds of several different legume species (Table 1). The minimum amount of inhibitor detectable under the conditions used was about 3-4 pg of soybean trypsin inhibitor, or its equivalent. The size of the inhibited areas was found to be time dependent, areas continuing to increase up to about 5 days diffusion at 4°C. but provided the standards
TABLE TR~PSIN
INHIBITOR
AND “ROCKET” ASSAY”
CONTENTS
OF VARICIUS
French bean (P/lu.srollr.\ L~ltl~1tri.c ) Garden pea ( Pi.\rrnr .\ll/il~/lm ) Soybean (G/.vc~im~ t?l(l.\. ) Winged bean (P.sophoc~crr~“r,s tl’lrtr,~l~ni~ll~hlt.s) Cowpea (L'ig:,ru rrtfguic~ultrftr) varieties TVu 2027 TVu 4557 TVu 76 TVu 3629 TVu 1190E TVu 57 TVu 1502.ID
1 MEAIS,
AS DETERMINED
BY DIFFUSION
ASSAY
ASSAY USING IMMOBILIZED TRYPSIN. AND BY SPECTROPHOTOMETRIC (INHIBITION ot TRYPSIN-CATALYZED HYDROLYSIS 0~ BAPNA) Units
Sample
SAMPLE
441
INHIBITORS
of inhibitorimg”
Percentage
Diffusion assay
Rocket assay
Spectrophotometric assay
6.2
6.9
6.4
0
0
26.5
30.7
24.5
10.9 7.4 4.9 4.5 3.2 2.4
98, 438-444
BIOCHLMISTRY
Two Simple
JOHN
A.
(1979)
Quantitative Assays for Trypsin Using Immobilized Trypsin GATEHOUSE
AND
ANGHARAD
Received
March
M. R.
Inhibitors
GATEHOUSE
7, 1979
Two new methods for quantitative assay of trypsin inhibitors, suitable for large numbers ofsamples, are described. The assay methods use trypsin-Sepharose conjugates incorporated into agarose gel slabs. Trypsin inhibitors are allowed to diffuse into, or are electrophoretically moved through, the slabs, and the consequent areas of inactivation of immobilized trypsin are visualized using a histochemical enzyme substrate. Quantitation of the trypsin inhibitor content of samples can be made on the basis of the inactivated areas. The limit of detection is 1-2 pg of soybean trypsin inhibitor and determinations are reproducible to 10% or better. Measured trypsin inhibitor contents of several legume species and varieties agree with spectrophotometric determinations.
Protein trypsin inhibitors are well known as potential antimetabolic components of many plant tissues, and as such their presence may be undesirable: for example, raw soybeans are antinutritional to animals due to, among other things, the high concentration of trypsin inhibitors they contain (1.2). On the other hand, in the native plant trypsin inhibitors may confer desirable properties; it has been hypothesized that trypsin inhibitors (as the major proteinase inhibitors) are responsible for resistance of plant material toward attack by insects (3-5) fungi, and bacteria (6,7). Recently it has been shown that an elevated level of trypsin inhibitors was responsible in the laboratory for the resistance of the seeds of a variety of cowpea (Vigrza ung:lriculatu) toward the bruchid beetle Cal1osobtwc~hu.s rnaculatuLs (8). Further, the antimetabolic properties of trypsin inhibitors may be removed by cooking or processing (9,10), and since the proteins as a rule have a high sulfur content (11) they may make a significant contribution to the nutritional value of legumes, which are deficient in sulfur amino acids (12,13), as animal or human foodstuffs. 0003.2697/79/140438-07$02.00/O Copyright All rights
I 1979 by Academc Press. Lnc. 01. reproductmn ,n any form reserved.
438
Irrespective of whether trypsin inhibitors are considered as desirable or undesirable components of plant materials, the levels at which they are present need to be estimated for quality control, or for comparisons between species and varieties. It is thus useful to have a quantitative assay for trypsin inhibitory content which can be applied to the large numbers of samples involved in screening programs. While a number of reasonably accurate methods are available for quantitative estimation of trypsin inhibitors, mainly by measuring the reduction in the rate of hydrolysis by trypsin of natural or artificial substrates caused by the inhibitor (14-16), they are time consuming. On the other hand, several rapid methods exist for the detection of trypsin inhibitors (17- 19) but none is quantitative. The present paper describes two simple, quick, quantitative methods for estimation of trypsin inhibitors based on diffusion or electrophoresis of the inhibitors into gels containing immobilized trypsin. The methods may be applied to small samples (10 mg of plant material or less) and are suitable for estimating the inhibitor content
QUANTITATION
OF TRYPSIN
of single seeds, or single cotyledons, with sufficient accuracy for plant breeding purposes. MATERIALS
AND METHODS
Muterids. Trypsin (Type I, from bovine pancreas), soybean trypsin inhibitor (Type l-S), and the enzyme substrates cu-Nbenzoyl - DL - arginine -p - nitroanilide-HCl (BAPNA)’ and a-N-benzoyl-DL-arginineP-naphthylamide- HCl (BAN A) were obtained from Sigma (London) Chemical Company Ltd., Poole, Dorset, England. Fast blue B salt (diazonium salt of o-dianisidine) and cyanogen bromide (pure) were obtained from Koch-Light Laboratories Ltd., Colnbrook, Buckinghamshire, England. Agarose, immunodiffusion plates, and well punches were purchased from Miles Laboratories Ltd., Slough, England, and the Sepharose 4B was supplied by Pharmacia Fine Chemicals, Uppsala, Sweden. Buffer components and reagents were obtained from British Drug Houses (BDH) Ltd., Poole, Dorset, England, and were of analytical grade when necessary. Merhods. Trypsin was immobilized by coupling onto cyanogen bromide-activated Sepharose 4B (approximately 2.5 mg protein/ml of gel). Both the activation and subsequent coupling were carried out according to the method of March et ~11.(20). Prior to use the coupled trypsin was diluted with distilled water (1:2) to form a slurry of known enzyme concentration. The assay gels were prepared by dissolving agarose in 1 vol of boiling water on a thermostatically controlled magnetic stirrer and then adding 1 vol of buffer to give a final concentration of 1% agarose in 0.05 M Tris-HCl, 0.02 M CaCl,, pH 8.2. The gel mixture was equilibrated at 55°C and the trypsin/Sepharose slurry (equilibrated at 55°C for 10 min) was added and mixed ’ Abbreviations used: arginine-p-nitroanilide-HCI; arginine-P-naphthylamide-HCI.
BAPNA. BANA,
a-N-benzoyl-ol.a-A’-benzoyl-oL-
439
INHIBITORS
thoroughly (25 ~1 of slurry per milliliter of 1% agarose was found to be optimum for the levels of trypsin inhibitor assayed). Gels were cast immediately. Diffusion gels of loml volume were cast in Miles immunodiffusion plates. Slab gels for electrophoresis were of 30-ml volume and were cast on oven-dried glass plates (110 x 205 mm) precoated with 0.5% agarose in 50% aqueous methanol. Trypsin inhibitor samples were routinely prepared by extracting 20-mg samples of meal in 500 ~1 of 0.05 M TrisHCl, 0.02 M CaCl, buffer, pH 8.2, for 4 h at room temperature. The extracts were centrifuged for 5 min in a bench centrifuge (9OOOg)and lo- or 20+1 samples of the supernatants were applied to wells punched through the gel using a 3- or 4-mmdiameter punch. Either cowpea trypsin inhibitor, purified by affinity chromatography (21), or soybean trypsin inhibitor standards were run on each gel. Diffusion plates were left 16 h at room temperature and then transferred to precoated agarose glassplates. Rocket electrophoresis was carried out on a flatbed electrophoresis apparatus with a water-cooled platen, using barbitone-sodium buffer, pH 8.6 (22). The gel was allowed to run overnight at 5 V/cm. Following electrophoresis or diffusion the gels were rinsed in distilled water and subsequently pressed and air-dried (23). They were then incubated with the histochemical substrate and stain (30 mg fast blue B salt dissolved in 30 ml sodium acetate (0.1 M, pH 6.5), 24 ml sodium chloride (0.14 M), and 3 ml potassium cyanide (0.02 M), to which was added 24 mg BANA dissolved in 3 ml methanol (24); the reagent must be used immediately). Maximum staining was achieved in approximately 30 min after which time the gels were again rinsed and finally pressed and airdried. RESULTS Sepharose-coupled trypsin was shown to be reasonably stable under the conditions used for incorporation into agarose gels by
440
GATEHOUSE
AND
GATEHOUSE
FIG. 1. Diffusion assays for trypsin inhibitors. Wells standards, 0, 2, 4. 6, 8. and 10 fig inhibitor, respectively. 10 Pg.
comparing the rates of BAPNA hydrolysis by the conjugate on incubation at 55°C and at room temperature; no significant difference was observed after 15 min. Further, trypsin activity could be detected in an agarose gel slab incorporating the Sepharose-trypsin conjugate by incubation in a solution of BAPNA, on which the solution rapidly turned yellow due to release of pnitroaniline. However, to locate the active trypsin a histochemical substrate, BANA, that gave an insoluble colored product on trypsin hydrolysis had to be employed. An agarose gel slab containing trypsin-sepharose was pressed, dried, and incubated with BANA-fast blue B stain solution. Trypsin activity could now be seen to be located at points, giving the agarose gel a speckled appearance like a poorly printed photograph. The red color produced by hydrolysis of BANA is fixed in the gel and will not wash out; the gel may thus be washed. pressed. and dried without altering the stained areas. The initial method devised for assay of trypsin inhibitors was by radial diffusion. If a solution of trypsin inhibitor is placed in a circular well punched in the agaroseSepharose- trypsin gel, the inhibitor will diffuse outward in all directions. As it encounters trypsin molecules, the inhibitor will bind to them and inactivate them. Since the trypsin-inhibitor binding is strong and
1. 2, 3. 4. 5, and 6: cowpea trypsin Wells 7 and 8: soybean trypsin
inhibitor inhibitor.
kinetically stable at the pH used (11) inactivation is effectively permanent and the inhibitor cannot diffuse away once bound. The unbound inhibitor will continue diffusing until all of it has complexed with trypsin, the result being a circular area around the well where the immobilized trypsin has bound inhibitor and been in1. 5
FIG. 2. Calibration of diffusion trypsin inhibitor (CPTI) and hibitor (SBTI) standards.
assays, using cowpea soybean trypsin in-
QUANTITATIiON
OF TRYPSIN
activated. If the agarose gel is now incubated with the histochemical trypsin substrate BANA the effect of the inactivation is seen as a clear area round the well where no trypsin-catalyzed hydrolysis has occurred (Fig. 1). The area of the circle is proportional to the amount of inhibitor originally placed in the well; the diameter of the circles may be measured with dividers and the areas calculated. Typical clalibrations using soybean trypsin inhibitor and cowpea trypsin inhibitor are given in Fig. 2. The unknown trypsin inhibitor content of samples may now be estimated from the standard curve. The reproducibility of this assay was tested using purified cowpea and soybean trypsin inhibitors: on the same
batch of slabs reproducibility is good and values may be estimated with an error of less than 10%. The standard curve, however, was found to vary slightly from one batch of experiments to another, due to nonreproducibility in making up the slurry of trypsin--Sepharose. The method is of general applicability, as shown by the estimations of inhibitor content of seeds of several different legume species (Table 1). The minimum amount of inhibitor detectable under the conditions used was about 3-4 pg of soybean trypsin inhibitor, or its equivalent. The size of the inhibited areas was found to be time dependent, areas continuing to increase up to about 5 days diffusion at 4°C. but provided the standards
TABLE TR~PSIN
INHIBITOR
AND “ROCKET” ASSAY”
CONTENTS
OF VARICIUS
French bean (P/lu.srollr.\ L~ltl~1tri.c ) Garden pea ( Pi.\rrnr .\ll/il~/lm ) Soybean (G/.vc~im~ t?l(l.\. ) Winged bean (P.sophoc~crr~“r,s tl’lrtr,~l~ni~ll~hlt.s) Cowpea (L'ig:,ru rrtfguic~ultrftr) varieties TVu 2027 TVu 4557 TVu 76 TVu 3629 TVu 1190E TVu 57 TVu 1502.ID
1 MEAIS,
AS DETERMINED
BY DIFFUSION
ASSAY
ASSAY USING IMMOBILIZED TRYPSIN. AND BY SPECTROPHOTOMETRIC (INHIBITION ot TRYPSIN-CATALYZED HYDROLYSIS 0~ BAPNA) Units
Sample
SAMPLE
441
INHIBITORS
of inhibitorimg”
Percentage
Diffusion assay
Rocket assay
Spectrophotometric assay
6.2
6.9
6.4
0
0
26.5
30.7
24.5
10.9 7.4 4.9 4.5 3.2 2.4
