J . Sci. Food Agric. 1984,35,987-995

Proteins and Trypsin Inhibitor Activity of Guar (Cyamopsis tetragonoloba L. Taub) Seed

Perminder Kaur and Iqbal S. Bhatia

Department of Biochemistry, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana 141 004, India

(Manuscript received 14 October 1983)

Proteins soluble in tris-acetate buffer (pH 9.0) were fractionated by DEAE- cellulose, DEAE-Sephadex A-50 and Sephadex G-200 column chromatography. The purified proteins which contained 5-6% carbohydrate, had molecular weights of 125 900 and 22 390 amu. The high molecular weight fraction was homogeneous by polyacrylamide gel electrophoresis. Proteins extracted in phosphate buffer (0.lh1, pH7.6) when subjected to Sephadex G-200 gel chromatography were resolved into three fractions, all of which showed considerable trypsin inhibitor activity. Germination for 3 days reduced the trypsin inhibitor activity of the seed by about 30%.

Keywords: Cyamopsis tetragonoloba; protein; proteolytic activity; trypsin inhibitor activity.

1. Introduction

Guar (Cyamopsis tetragonoloba L. Taub) seed is a major source of a gum, a galactomannan, which has several industrial applications. The cotyledonous fraction remaining after the extraction of guar gum is a rich source of protein (38-55%) and is used in animal and poultry

The salt-soluble proteins of guar meal have been partially ~haracterised~ and the major globulin has been investigated in some detaiL4 Despite its high protein content, guar meal has been reported to retard growth and even cause mortality in This effect has been variously attributed to the presence of trypsin inhibitor^,^,' haemagglutinins' and saponins" in the meal. The present paper deals with the extraction and characterisation of the proteins, and the proteolytic and trypsin inhibitor activity of guar meal.

Experimental 2.1. Preparation of guar meal Guar seeds (cv. FS 277) were first crushed coarsely in a hand-operated grinder to remove the seed coat with the adhering gum. The cotyledons were then ground finely in a mortar and partially defatted with n-hexane (50 ml g-l) at ambient temperature for 48 h. The defatted meal contained, on average, 55% protein (Nx6.25).

2.2. Extraction of proteins from guar meal In preliminary experiments using several different buffers, it was found that as the pH of the extractant was lowered from 9.0 to 5.7, the amount of protein extracted decreased (Table 1).

Tris-acetate buffer ( 0 . 0 5 ~ , pH 9.0), which extracted most protein from the defatted guar meal, was chosen for subsequent column chromatographic separations: guar meal (3 g) was extracted twice with buffer (30ml) for 4 h at 4°C with continuous stirring, The supernatants obtained by centrifuging (120OOxg, 20min, 4°C) were pooled and dialysed against tris buffer for a total of 72 h with repeated changes.

987

988 P. Kaur and 1. S. Bhatia

Table 1. Protein and carbohydrate contents (expressed as percentage of guar meal dry weight) of dialysed extracts in different buffer solutions

Carbohydrate Protein as glucose

(Nx6.25) equivalents Extracting buffer (%) (%)

Tris acetate ( 0 . 0 5 ~ , pH 9.0) 39.6 5.85

Sodium phosphate ( 0 . 0 5 ~ . pH 6.7) 16.6 5.00 Sodium phosphate" (0 .1~ . pH 7.6) 21.4 5.60

Citrate-phosphateb ( 0 . 0 2 5 ~ ~ pH 5.7) 14.7 4.00

Sodium dihydrogen phosphate and disodium hydrogen phosphate." *Citric acid and disodium hydrogen phosphate.'*

Phosphate buffers,".'2 pH 7.6, 6.7 and 5.7, were used as protein extractants for studying the action of trypsin, leucineaminopeptidase and caseolytic enzymes respectively, these pH values corresponding to the pH optima of the enzyme. Details of protein extraction are given under individual enzyme assays.

2.3. Chromatographic separation of tris-acetate soluble proteins

2.3.1. DEAE-cellulose chromatography DEAE-cellulose was washed according to the procedure of Pe te r~on , '~ equilibrated with sodium phosphate buffer ( 0 . 0 1 ~ , pH 8.0) and packed into water jacketed column (2.5x18cm) maintained at 4°C. Dialysed tris-acetate extract (containing 0.6-0.9 g protein) was loaded and eluted with the same buffer, but with a gradient of increasing sodium chloride concentration (0, 0.025, 1 ~ ) . Fractions (10 ml) were collected and analysed for proteinI4 and carbohydrate" content. The fractions from each of the three protein-rich peaks found were pooled (Al, A2 and A3 respectively).

2.3.2. DEAE-Sephadex A-50 chromatography of fraction A3 Fraction A3, which contained most protein, was precipitated with saturated (NH4)2 SO4 solution and dialysed for 48h against phosphate buffer ( 0 . 0 1 ~ . pH 8.0). The diaiysate (containing 0.3-0.4g protein) was applied to a column (2.5X25 cm) of DEAE-Sephadex A-SO equilibrated with sodium phosphate buffer ( 0 . 0 1 ~ , pH 8.0). The same buffer, but with increasing concentrations of NaC1: (0, 0.025, 0.05. 0.1, 0.2, 0.3, 0.4 and 0 . 5 ~ ) was used for elution, and lOml fractions were collected and analysed for protein and carbohydrate content. Of the four fractions obtained ( B l , BZ, B3 and B4), only B2 was obtained in sufficient quantity for further studies.

2.3.3. Sephadex C-200 chromatography of fraction B2, The fraction B2 (containing 8 k 9 0 mg protein) which was concentrated by dialysis against polyethylene glycol (mol. wt 20000) was fractionated on a column (2.5x53cm) of Sephadex G-200 gel equilibrated with sodium phosphate buffer (0.01h1, pH 8.0) containing 0 . 3 ~ NaCI. Elution was carried out with the same buffer containing 0 . 3 ~ NaCI. Fractions (3 ml) were collected and analysed for protein and carbohydrate content: two protein-rich peaks were found, and the respective fractions were pooled (C, and C?).

The void volume (Vo) of the column was determined using 0.03% solution of blue dextran. A solution containing catalase, bovine serum albumin, pepsin and ribonuclease was used to prepare a standard molecular weight curve.

2.4. Chromatography on Sephadex G-200 of guar meal protein extracted with phosphate buffer Proteins were extracted with phosphate buffer ( O . ~ M , pH 7.6) as described in section 2.2. Dialysed protein extract (containing 50-60 mg protein) was chromatographed on Sephadex G-200

Proteins and TIA of guar seed 989

column (2.5x66cm) using the same buffer as eluent. Fractions (3ml) were collected and analysed for protein content. Three peaks were obtained (Fl, F2 and F3) and tested for trypsin inhibitor, caseolytic and leucineaminopeptidase activities.

2.5. Polyacrylamide gel electrophoresis (PAGE) of protein extracts and fractions Polyacrylamide gels (7.5%) in tns-glycine buffer (pH 8.3) were run at 18OV for 2.5 h using Systronics (India) apparatus'". The gels were stained with Coomassie brilliant blue solution (0.1%) in dilute acetic acid (7%).

2.6. Proteolytic activity of guar meal and its protein fractions The proteolytic activity was determined using casein and leucine-p-nitroanilide as the substrates according to the method of Chrispeels and Boulter".

2.7. Caseolytic activity of guar meal and its protein fractions Guar protease extract was prepared by stirring guar meal ( lg) with 15ml citrate-phosphate buffer (25 mM, pH 5.7) for 2 h at room temperature. The protein contents of both the extract and fractions F1, F2 and F3, which had been dialysed against the buffer at pH 5.7, were determined. The assay mixture containing casein (lml 1% wt/vol solution Hammersten quality in 25mM citrate phosphate buffer pH 5.7), 0.2 ml protease extract or fraction, and 0.8 ml water was incubated in a water bath (45"C, 1 h). The reaction was stopped by adding 1 ml of 15% TCA. The solubilised protein was determined by the method of Lowry et

2.8. Leucineaminopeptidase (LAPase) activity of guar meal and its protein fractions Guar meal ( lg) was extracted with 15ml of phosphate buffer ( 5 0 m ~ , pH 6.7) for 2 h with stirring. The protein content of both the supernatant and fractions F1, F2 and F3 which had been dialysed against the buffer at pH 6.7 was determined. Leucine-p-nitroanilide (25 mg) was dissolved in dimethylsulphoxide (lml) and made up to 50ml with phosphate buffer (SOmM, pH 6.7). Leucine-p-nitroanilide solution (2ml) was incubated with 0.1 ml of guar meal extract or fraction (30"C, 20 min). The reaction was stopped by adding 1 ml of 1~ perchloric acid. A blank was prepared by precipitating the enzyme with 1~ perchloric acid before adding the substrate. The contents were filtered using Whatman No. 42 paper and the absorbance read at 410nm against the blank. One unit of activity corresponds to a change of 0.01 absorbance unit per 20 min.

Table 2. Protein and carbohydrate contents of fractions separated by DEAE-cellulose, DEAE- Sephadex A-50 and Sephadex G-200 column chromatography (expressed as percent volume)

Protein recovered

Fraction (%)

Carbohydrate" as glucose

Proteina equivalents (%) (%)

DEAE-cellulose column A, 1.4 A2 1.3 A3 97.3

DEA E-Sephadex A-SO column (A,) Bl 3.3 B2 40.4 B3 29.2 B4 27.1

Sephadex G-200 column (B2) c1 45.0 c2 55.0

33.3 66.1 91.4 8.6 93.1 6.9

96.0 4.0 95.7 4.3 91.9 2.1 98.1 1.9

95.3 4.1 94.2 5.8

Carbohydrate (mg 100 mg-'

protein)

200.0 9.4 7.4

4.2 4.6 2.1 1.9

4.9 6.2

Mol. wt

125900 22 390

"The values refer to the content in l00ml of the fraction calculated as [C/(P+C)]X100 and the % protein calculated by difference. P and C refer to protein and carbohydrate content, respectively.

990 P. Kaur and I. S. Bhatia

2.9. Trypsin inhibitor activity Guar meal (1 g) was extracted with 20ml of phosphate buffer (0.lh1, pH 7.6) with stirring (1 h) and the clear supernatant was diluted to 50ml with the same buffer. The protein content of the extract was determined by the method of Lowry et u1.l4. The proteins of guar meal extract before and after heat treatment (95"C, 1 h) and of guar seed after germination (at 22 f 1.C) were tested for trypsin inhibitor activity by the method of Kakade et u1.'* using a-N-benzoyl-DL-arginine-p- nitroanilide (BAPA) as substrate. The concentration of trypsin used was 2 mg 100 ml-'. Fractions F1, F2 and F3 were also tested for trypsin inhibitor using BAPA as substrate.

2.10. Germination Guar seeds were steeped in water for 24h and were allowed to germinate in Petri dishes lined with moist filter papers (22"Ck 1). The cotyledons (1 g) were removed at 1-day intervals and ground using a cold mortar and pestle and a total volume of 15ml of citrate-phosphate buffer ( 2 5 m ~ , pH 5.7), phosphate buffer ( 5 0 m ~ , pH 6.7) and phosphate buffer ( 0 . l ~ , pH 7.6). The extracts were centrifuged (12 000 X g, 20 min), dialysed against the respective buffers, and used for the assay of caseolytic, leucineaminopeptidase and trypsin inhibitor activities, respectively.

3. Results and discussion 3.1. Fractionation and characterisation of proteins extracted by tris-acetate buffer The third fraction obtained by DEAE-cellulose chromatography which eluted as a single peak, A3, accounted for 97% of the total protein recovered (Table 2). This fraction was further

Figure 1. a, Polyacrylamide gel electrophoresis (PAGE) of guar proteins extracted in tris-acetate buffer (pH 9.0); b and c, PAGE of purified protein fractions C1 and Cz, respectively; d, e and f , PAGE of proteins extracted in phosphate buffers of pH 7.6, 6.7 and 5.7, respectively.

Proteins and TIA of guar seed 991

resolved by column chromatography on DEAE-Sephadex A-50; 40% of the protein appeared in the second fraction Bz. Chromatography of Bz on Sephadex G-200 column resulted in two fractions, C1 and C,, of approximately equal protein content and having molecular weights 125900 and 22390 amu, respectively. Nath et aL4 working on the fractionation of guar meal proteins reported that the major globulin (mol. wt 223000) consisted of six sub-units ranging from 25 000 to 52 000 amu. In the present study the molecular weight of C1 was found to be six times that of C,. Because the high molecular weight fraction (Cl) eluted close to the void volume of Sephadex G-200 gel, aggregation of components is suggested. Aggregates of soya bean proteins eluting near the void volume on Sephadex G-200 column have recently been reported by wanglg.

Fraction C1 and C2 were associated with 5-6% carbohydrate, and may justifiably be considered to be glycoproteins. There was a poor correlation between the carbohydrate and protein peaks, the earlier fractions being richer in carbohydrate than the later ones. Although the protein fractions eluted as single peaks, they exhibited microheterogeneity similar to that recently reported by Lamport" in glycoprotein molecules.

Polyacrylamide gel electrophoresis (PAGE) of tris-acetate extract resulted in ten polypeptide bands (Figure 1, a). Fraction C1 gave a single diffused band, but C2 two closely placed bands (Figure 1, b and c respectively). There is also a trace of a band in the high mol. wt region (Figure

Table 3. Trypsin inhibitor activity (TIA) of guar meal extract in phosphate buffer (0.1h1, pH 7.6)

Guar meal Absorbance extract Protein (410nm) TU TIU mg-' Inhibition

(mu 01 9) (A) (AX100) TIU protein (%)

0.0 - 0.235 23.5 - - -

0.5 90.9 0.215 21.5 2.0 22.0 9.0 1.0 181.8 0.205 20.5 3.5 19.3 14.9 1.5 272.7 0.190 19.0 4.5 16.5 19.1 2.0 363.7 0.170 17.0 6.5 17.8 27.6

Extrapolated value=24.5 TIU mg-' protein. TU=Trypsin units; TIU=Trypsin inhibitor units.

Table 4. TIA of fractions F,, Fz and F3 separated on a Sephadex G-200 column from a guar meal extract in phosphate buffer (O.~M, pH 7.6)

Protein Absorbance fraction Protein (410nm) TU TIU Inhibition

(ml) OCg) (A) (Ax100) TIU mg-' protein (%)

Frnclion F, - - - 0.0 - 0.245 24.5

0.5 172.0 0.190 19.0 5.5 31.9 22.4 1 .o 344.0 0.220 22.0 2.5 7.3 10.2

Extrapolated vaIue=55 TIU mg-' protein Fraction F2

0.5 160.0 0.200 20.0 4.5 28.1 18.4 1 .o 320.0 0.215 21.5 3.0 9.4 12.2

Extrapolated value=46 TIU mg-' protein Fraction F3

- - 0.0 - 0.245 24.5 -

0.2 83.2 0.170 17.0 7.5 90.0 30.6 0.3 124.8 0.160 16.0 8.5 68.0 34.7 0.4 166.4 0.150 15.0 9.5 57.0 38.8 0.5 208.0 0.155 15.5 9.0 43.0 36.7 0.6 249.6 0.155 15.5 9.0 36.0 36.7 1.0 416.0 0.145 14.5 10.0 24.0 40.8

Extrapolated value=100 TIU mg-' protein

TU=Trypsin units; TlU=Trypsin inhibitor units.

992 P. Kaur and I. S . Bhatia

1, c). This was unexpected and may possibly be an artefact resulting from association of low mol. wt proteins. These results contrast with those of Nath et u I . ~ who reported the presence of only three bands when a IM NaCl extract of guar proteins was subjected to PAGE; two being of high and one of low mol. wt. Fraction C1 may correspond to one of these high mol. wt and C2 to the low mol. wt, polypeptides. PAGE of proteins extracted in phosphate buffer at pH 7.6 gave one high mol. wt and two low mol. wt bands (Figure 1, d). The proteins extracted in phosphate buffer at pH 6.7 and 5.7 both gave two diffused bands of low rnol. wt (Figure 1, e and f).

3.2. Trypsin inhibitor activity (TTA) of proteins extracted by phosphate buffer ( O , ~ M , pH 7.6) Preliminary studies with a crude extract indicated the presence of a non-dialysable factor with a low TIA of 24.5 TIU mg-l protein (Table 3). Of the fractions tested, F3 had the maximum TIA of about 100 TIU mg-I protein, followed by F1 and F2 with 55 and 46 TTU mg-' protein, respectively (Table 4). The lower TIA values of the crude extract compared to those of the fractions may indicate an in-vivo association of the individual proteins with other components resulting in a decreased inhibitory effect.

Experiments were performed to check whether the inhibitor could be destroyed by heat or germination. Heating the crude extract (95"C, 1 h) had no apparent effect on the inhibitor: the observed TIU/mg-' protein being 24.5 and 26.5 for unheated and heated extracts respectively (Tables 3 and 5 ) . This agrees with the results of earlier workers8.

Germination led to some decrease in TIU values initially, but extending the germination period to 3 days did not cause further change (Table 6 and Figure 2). The results, expressed per

Table 5, Trypsin inhibitor activity (TIA) of guar meal extract in phosphate buffer (O.~M, pH 7.6) as influenced by heating (95°C 1 h)

Guar meal extract (mu ~- 0.0 0.5 1 .o 1.5 2.0

Absorbance Protein (410nm) TU

old (A) (Ax100)

- 0.235 23.5 43.3 0.225 22.5 86.7 0.210 21.0

130.0 0.215 21.5 173.4 0.215 21.5

~. TIU

~- -

1 .o 2.5 2.0 2.0

TIU mg-' Inhibition protein (%a) ~ _ _ _ _ ~

- - 23.1 4.3 28.8 10.6 15.4 8.5 11.5 8.5

Extrapolated value=26.5 TIU mg-' protein. TU=Trypsin units; TIU=Trypsin inhibitor units.

Table 6. Effect of germination on the trypsin inhibiting activity (TIA) of guar seed

Guar Absorbance Germination Moisture cxtract Protein (410nm) TU TIU mg-' Inhibition

(days) (%a) (ml) Wg) (A) (AX100) TIU protein (%) - - - I 61.9 0.0 - 0.235 23.5

0.5 86.7 0.230 23.0 0.5 5.8 2.1 1 .o 173.3 0.210 21 .0 2.5 14.4 10.6 1.5 260.0 0.200 20.0 3.5 13.5 14.9

0.5 66.7 0.215 21.5 0.0 1 .0 133.3 0.198 19.8 1.7 12.8 7.9 1.5 200.0 0.180 18.0 3.5 17.5 16.3 2.0 266.7 0.178 17.8 3.7 13.9 17.2

0.5 61.5 0.220 22.0 0.0 1 .o 123.1 0.215 21.5 0.5 4.1 2.3 1.5 184.6 0.190 19.0 3.0 16.3 13.6 2.0 246.2 0.190 19.0 3.0 12.2 13.6

- - - 2 67.9 0.0 - 0.215 21.5 - -

- - - 3 70.3 0.0 - 0.220 22.0 - -

TU=Trypsin units; TIU =Trypsin inhibitor units.

Proteins and TIA of guar seed

2ol 993

c 0

Figure 2. Effect of germination on the trypsin inhibitor activity of guar seed. 0, 1 day; 0, 2 days; 0, 3 days.

0 4

I I I 1.0 1.5 2 . 0

s" 0 . 5

Extract (mL)

mg protein in the extract, gave almost similar TIU values: the overall decrease in TIU was 3040%. Similar results have been reported with soya bean21, but there are other, conflicting, report^*^-^^.

3.3. Caseolytic activity (CA) of proteins extracted by citrate phosphate buffer (0.025111, pH 5.7) Guar meal extracted in this buffer gave 4.1 units g-' caseolytic activity (Table 7) compared with a reported value of 8.5 and 5.6 Kunitz units mg-' protein when the meal was extracted in the same buffer and IM NaCl, re~pectively.~ D'Souzag, on the other hand, reported the total absence of activity in guar meal. Fractions F1 and F3 had no measurable CA, while F2 had an activity of 7.9 units mg-' protein (Table 8). Nath et u Z . ~ also reported that, of the three fractions obtained by Sephadex G-200 gel chromatography of an NaCl extract of guar meal, only the third fraction had a measurable activity (11.4 caseolytic units mg-' protein).

3.4. Leucineaminopeptidase (LAPase) activity The guar meal proteins extracted in phosphate buffer (0.05h1, pH 6.7) had an activity of 44.5 LAPase units g-' (Table 7). This is the first report of LAPase activity in guar. Of the fractions F1, F2 and F3, only F2 had any activity-15.7 LAPase units mg-' protein (Table 8).

Table 7. Effect of germination on the caseolytic and leucineaminopeptidase (LAPase) activities of guar seed cotyledon ___~ ~~~

Caseolytic activity LAPase activity ~. . .~

Absorbance Caseolytic Absorbance LAPase Germination Moisture Enzyme (520 nm) units Enzyme (410 nm) units

(days) (%) (mU (A) (AX 100) (mu (A) (Ax100)

0 9.4 0.2 0.041 4.1 0.1 0.445 44.5 1 61.9 0.2 0.091 9.1 0.1 0.759 75.9 2 67.9 0.2 0.119 11.9 0.1 0.789 78.9 3 70.4 0.2 0.112 11.2 0.1 0.710 71.0 4 73.5 0.2 0.139 13.9 0.1 0.637 63.7 5 74.0 0.2 0.106 10.6 0.1 0.b24 62.4

994 P. Kaur and 1. S. Bhatia

Table 8. Caseolytic and leucineaminopeptidase (LAPase) activity of Sephadex G-200 gel separated fractions of guar meal extract in phosphate buffer ( 0 . h pH 7.6)

Caseolytic activity

Absorbance Caseolytic Caseolytic Protein Protein (520 nm) units units mg-' fraction old (A) ( A x 100) protein

- F1 262 0.00 0.0

F3 F2 254 0.02 2.0 7.87

200 0.00 0.0 -

LAPase activity

Absorbance LAPase LAPase (410 nm) units units mg-'

(A) (Ax 100) protein ~~ ~ ~

- 0.00 0 0 0.04 4.0 15.7 0.00 0.0 -

It was observed that the fraction F2 which had the lowest trypsin inhibitor activity (Table 4) had the maximum CA and LAPase activities (Table 8). The reverse was observed with fractions F1 and F3 indicating a reciprocal relationship between TIA and proteolytic activity,

3.5. Effect of germination on CA, LAPase and TI activities During germination, the caseolytic activity of the cotyledons initially increased up to the fourth day and then declined (Table 7) . This agrees with similar observations on other legumes.2c29

The LAPase activity increased steeply up to the second day of germination and then declined gradually (Table 7). It is apparent that the role of LAPase in the degradation of reserve proteins is confined only to the early phases of germination. A similar decrease in LAPase activity during the later stages of germination has also been reported in the cotyledons of peas and kidney

With the available data, it is not possible to conclude whether the observed increase in the CA and LAPase activities during germination is due to de-novo synthesis, activation of pre-existing enzymes or to the disappearance of one or more inhibitors. However, endogenous proteinase inhibitors are known to participate in the control of proteolysis," and it may be that on germination, these inhibitors are hydrolised and hence, rendered less effective.

beans.26.27. 30

4. Conclusions

The results presented here emphasise the complex nature of guar seed proteins. The association of carbohydrate moieties with the various polypeptide fractions suggests the predominance of glycoproteins in guar seed. Information about the level of proteolytic inhibitor activity associated with the protein fractions may be useful to plant breeders in selecting varieties with minimum levels of undesirable constituents.

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