Determination of amylase activity in cotyledons of Phaseolus vulgaris L. cv. carioca

Determination of amylase activity in cotyledons of Phaseolus vulgaris L. cv. carioca

Glaucia Almeida de MoraisI^*; Massanori TakakiII^*

^IUEMS - Cassilândia - MS - Brasil

^IIDepartamento de Botânica - UNESP - C. P. 199 - CEP 13506 -900 Rio Claro - SP - Brasil

ABSTRACT

Determination of α- and β-amylase activity in the extracts of cotyledons of Phaseolus vulgaris L. cv. cariocawas done using selective inactivation of α-amylase by lowering the pH of the incubation medium or by the use of EDTA as inhibitor or selective inactivation of β-amylase by the use of HgCl₂ or by heating to 70^ºC in the presence of CaCl₂; and still by using the reagent starch azure for specific determination of α-amylase. Results indicated that the methods used were inappropriate in this case, being indicated the determination of total amylase activity.

Key-words: α-amylase, β-amylase, determination, selective inactivation, Phaseolus vulgaris.

INTRODUCTION

Determination of the quantities of a- and β-amylase in the total amylase activity can be done by selective inactivation of β-amylase by heating the crude extract to 70^ºC or by the use of mercuric chloride, and inactivation of α-amylase by incubation of crude extract with EDTA, or by carrying out assay at low pH. These procedures were adopted by several authors, e.g. TÁRRAGO & NICOLÁS (1976), MONERRI & GUARDIOLA (1986) and CHRISPEELS & VARNER (1967).

DOEHLERT & DUKE (1983) and HIRASAWA (1989) used starch azure which allowed the quantification of enzymatic activity as a function of the color intensity, produced by the release of soluble fragments of starch linked to the Remazol Brilliant Blue dye as a result of the action of α-amylase.

In this work, we present results of tests conducted in accordance with the above procedures with the aim of showing the difficulties in the choice of a assured analysis of the contribution of α- and β-amylase for the total amylase activity in cotyledonsof Phaseolus vulgaris L. cv. carioca.

MATERIAL AND METHODS

Crude extracts (CE) were prepared according to the method of JULIANO & VARNER (1969). Cotyledons of Phaseolus vulgaris L. cv. carioca were ground in cold tris-maleate buffer (0.05M; pH 7.0), with the aid of a mortar and pestle. The homogenate was centrifuged for 20 min at 2.400 x g and the supernatant was considered as CE.

Amylase assays were done as follows:

(a) With the use of inhibitors. For the assay of total, α- and β-amylase the modified method of CHRISPEELS & VARNER (1967)was used. Reaction mixture contained 0.5 ml CE with 0.5 ml 40mM EDTA for α-amylase, 0.5 ml CE with 0.5 ml 0.4mM HgCl₂ for β-amylase or 1.0 ml CE for total amylase activity (CE was diluted with distilled water). To this, 1.0 ml starch solution (150 mg soluble starch, 600 mg KH₂PO₄ and 200 µmol CaCl₂ in 100 ml distilled water) was added and the reaction was carried for 5 min at 30^ºC. Reaction was stopped by adding 1.0 ml iodine reagent (3 mg de KI + 0.3 mg of I in 0.05N HCl). For the reading, the mixture was diluted with distilled water up to 13.0 ml and the absorbance read at 620nm in a spectrophotometer Spectronic 20D.

One unit of enzyme was considered as the quantity that causes an alteration of 0.1 in absorbance.

(b) β-amylase assay with inactivation of α-amylase by lowering the pH of the incubation medium. A modified method of MONERRI & GUARDIOLA (1986) was used which was similar as above, except that the starch solution was prepared in citrate-phosphate buffer, pH 3.0. The reaction was carried out and stopped as above.

(c) α-amylase assay with inactivation of β-amylase by heating to 70^ºC. This was done according to the method of TÁRRAGO & NICOLÁS, 1976. Aliquot of 5.0 ml of CE with 3, 6, or 9mM CaCl₂ were heated in water-bath at 70^ºC for 5, 10 or 15 min. After this treatment, the assay followed the procedure as described for the total amylase activity.

(d) α-amylase assay with starch azure reagent.

Two methods were tested. According to the first method of Hirasawa (1989) 2ml of 2% starch azure (w/v) were slowly heated before the assay until boiling. To this solution, 1 ml of sodium acetate buffer 0.2M (pH 5.4), 0.12 ml of CaCl₂ 0.1M 0.83 ml of deionized water and 50 µl of CE were added. The reaction was carried out for 5 min at 37^ºC under agitation and was stopped with addition of 1 ml of 50% trichloroacetic acid (TCA) (w/v). The resultant mixture was centrifuged at 1000 x g for 20 min, filtered and absorbance was read at 590nm.

According to the second method (Doehlert & Duke,1983) a solution of 2% starch azure (w/v) was prepared in100mM potassium succinate (pH 6.0) with 3mM CaCl₂. This solution was slowly heated. After boiling, the solution was stirred continuously until use. The reaction mixture composed of 5 ml of the starch azure solution (placed in a test tube in a water-bath at 30^ºC) plus 1.0 ml of CE. In time designated as "zero", an aliquot of 1.0 ml was removed and placed in a test tube with 1.0 ml of 20% TCA (w/v). Additional aliquots were taken in times settled for the assay. After centrifugation, the absorbance of supernatant was read at 590 nm.

Protein determination: Protein concentrations were estimated according to LOWRY et al. (1951), using bovine serum-albumine as a standard.

Electrophoresis: Electrophoretic runs, for visualizing the enzymes present in the CE's of the different treatments, were done according to the method of LAEMMLI (1970), with modifications described in Table 1 and the exclusion of dodecyl sodium sulfate (SDS). The system comprises a 3% stacking gel (1.0 cm length) and 7.5% separation gel (6.0 cm length).

Running conditions: Electrophoretic runs were done in 80V constant voltage in a vertical slab, for about 2 hr.

Running buffer (pH 8.25): 3g of Tris + 14.4 g of glycine.

Visualization: After running, gels were incubated at 30^ºC in a 0.5% starch solution in 0.1M acetate buffer (pH 5.0) for 30 min and, thereafter, washed and transferred to iodine solution for about one min. After this time, a new washing of the gels was conducted. Bands with amylolytic activity appeared brightly while the rest of the gel was colored as a dark blue (JACOBSEN, SCANDALIOS & VARNER, 1970and TÁRRAGO & NICOLÁS, 1976).

The procedure for the preparation of the samples is described in Table 2. An aliquot of 30ml was used for each gel well. Samples submitted to inhibitors of amylase activity were kept incubating for 1 hr before the addition of the buffer in the sample.

RESULTS AND DISCUSSION

According to DOEHLERT & DUKE (1983), inactivation of β-amylase through heating was described in barley, where the α-amylase was a stable enzyme in this condition. Our results indicated a reduction of α-amylase activity, mainly in extracts containing only 3mM CaCl₂ and after 15 minutes heating complete inactivation of the enzyme was observed (Figures 1 and 2). Heating the extract for 15 minutes with 9mM CaCl₂ maintained the activity at the same level of 5 min but lower than the control activity determined after selective inhibition by HgCl₂.

There are many other reports on the use of the heating method, e.g.by ADAMS et al. (1980) on seeds of Glycine max, PRISCO et al. (1981) on cotyledons of Vigna unguiculata, CHIBA et al. (1990) on Oryza sativa, and AKAEVA & FURSOV (1990) on Triticum aestivum. In all cases, concentration of CaCl₂ was fixed allowed the stability of α-amylase during the heating time used. MAC GREGOR et al. (1971), using ionic exchange chromatography, corroborated that treatments of heating to 70^ºC for 15 minutes completely inactivated the β-amylase in barley extracts, without altering the α-amylase properties.

BATTERSHELL & HENRY (1990), verified that the heating of wheat seeds extracts at 70^ºC for 15 min decreased α-amylase activity by more than 80%. These authors explained on the basis of a possible difference in the enzyme structure or in the absence of a α-amylase inhibitor as the one that existed in barley, which stabilized the enzyme during heating. In Zea mays, WARNER et al. (1991) related the complete loss of α-amylase activity by heating to 70^ºC for 15 minutes in the presence of 10mM CaCl₂.

It was interesting to note that values obtained for a-, and β-amylase activities with inhibitors (EDTA and HgCl₂) together exceeded the total amylase activity (^{cf. figure 3}).

ROBBINS & EGAN (1992) reported that mercuric chloride was not effective in the selective inhibition of β-amylase in sorghum, although TÁRRAGO & NICOLÁS (1976) used successfully 5mM EDTA and 5mM HgCl₂ for the differentiation between α- and β-amylase in cotyledons of Lens sp.

KOSHIBA & MINAMIKAWA (1981) presented data showing the inactivation of α-amylase by EDTA in cotyledons of Vigna mungo, while NORBY & RINNE (1985) reported that treatment with EDTA did not inactivate completely in cotyledons of Glycine max.

The results obtained when the β-amylase activity was estimated by submitting the CE to a substrate prepared in pH 3.0 are presented in ^{Figure 3}. This procedure, according to MONERRI & GUARDIOLA (1986) would be preferable to the use of inhibitors, since the EDTA would lead to an incomplete inactivation, superestimating β-amylase activity. However, it was verified that the sum of α- and β-amylase does not correspond always to the total amylase activity.

The two methods tested here with the starch azure were also inadequate, since the quantification of the reaction was impossible due to a high viscosity of the reagent and the coefficients of variation exceeding the 10% limit settled for assays with inhibitors. The pattern obtained in these assays did not follow the one obtained for the total amylase activity with methodologies used previously (Figure 4).

Based on the use of inhibitors (EDTA and HgCl₂) and in the procedure of heating to 70^ºC, it was verified that two bands (1 and 2) showed characteristics of α-amylase (inhibition by EDTA, stability under heating to 70^ºC in the presence of 9mM CaCl₂) and two (5 and 6) of β-amylase (inhibition by HgCl₂, sensibility to heating). Bands 3 and 4 did not allow a well-defined characterization, since they were inhibited by the several procedures (figure 2).

As a conclusion, we suggest the determination of total amylase activity, without distinction between α- and β-amylase in the assays with cotyledons of P. vulgaris L. cv. carioca, with methodologies tested in the present work.

REFERENCES

ADAMS, C.A.; RINNE, R.W.; FJERSTAD, M.C. Starch deposition and carbohydrase activities in developing and germinating soya bean seeds. Annals of Botany, 45:577-582, 1980.

AKAEVA, M.M.; FURSOV, O.V. Synthesis, activation, and secretion of α-amylase in the aleurone layer and scutellum of the wheat caryopsis. Fiziologiya Rastenii, 37:1180-1185, 1990.

BATTERSHELL, V.G.; HENRY, R.J. High-performance liquid chromatography of α-amylases from germinating wheat and complexes with the α-amylase inhibitor from barley. Journal of Cereal Science, 12:73-81, 1990.

CHIBA, Y.; UI, M.; KATO Y.; NAKAJIMA, T.; ICHISIMA, E. Two groups of intracellular α-amylase isoenzymes from cultured rice cells. Phytochemistry, 29:2075-2078, 1990.

CHRISPEELS, M.J.; VARNER, J.E. Gibberellic acid-enhanced synthesis and release of α-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiology, 42:398-406, 1967.

DOEHLERT,D.C.; DUKE, S.H. Specific determination of α-amylase activity in crude plant extracts containing β-amylase. Plant Physiology, 71:229-234,1983.

DRUMM, H.; ELCHINGER, I.; MÖLLER, J.; PETER, K.; MOHR, H. Induction of amylase in mustard seedlings by phytochrome. Planta, 99:265-274,1971.

HIRASAWA, E. Auxins induce α-amylase activity in pea cotyledons. Plant Physiology, 91:484-486, 1989.

JACOBSEN, J.V., SCANDALIOS, J.G.; VARNER, J.E. Multiple forms of amylase induced by gibberellic acid in isolated aleurone layers. Plant Physiology, 45:367-371, 1970.

JULIANO, B.O.; VARNER, J.E. Enzymic degradation of starch granules in the cotyledons of germinating peas. Plant Physiology, 44:886-892, 1969.

KOSHIBA, T.; MINAMIKAWA, T. Purification by affinity chromatography of germinating Vigna mungo seeds. Plant & Cell Physiology, 22:979-987, 1981.

LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature, 4:680-685, 1970.

LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L.; RANDALL, R.J.. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193:265-275, 1951.

MACGREGOR, A.W.; LABERGE, D.E.; MEREDITH, W.O.S. Separation of α-amylase and β-amylase enzymes from barley malt by ion-exchange chromatography. Cereal Chemistry, 48:490-498, 1971.

MONERRI, C.; GUARDIOLA, J.L. Las amilasas del guisante. Su estimación en extractos crudos. Revista de Agroquímica y Tecnologia de Alimentos, 26:219-233, 1986.

NORBY, S.W.; RINNE, R.W. Further studies on the electrophoretic patterns of the multiple forms of amylase in soybean seeds. Crop Science, 25:990-994, 1985.

PRISCO, J.T.; ENEAS FILHO, J.; GOMES FILHO, E. Effect of NaCl salinity on cotyledon starch mobilization during germination of Vigna unguiculata (L.) Walp seeds. Revista brasileira de Botânica, 4:63-71, 1981.

ROBBINS, D.J.; EGAN, B. Failure of mercuric chloride to selectively inhibit β-amylase in sorghum malt. Journal of Institute of Brewing, 98, 383-385, 1992.

TÁRRAGO, J.F.; NICOLÁS, G. Starch degradation in the cotyledons of germinating lentils. Plant Physiology, 58:618-621, 1976.

WARNER, D.A.; GROVE, M.J.; KNUTSON, C.A. Isolation and characterization of α-amylases from endosperm of germinating maize. Cereal Chemistry, 68:383-390, 1991.

Received: 23 September 1997;

Revised: 26 May 1998;

Accepted: 20 July 1998.

*

correponding author

Publication Dates

• Publication in this collection
  21 Sept 2011
• Date of issue
  June 1998

History

• Accepted
  20 July 1998
• Received
  23 Sept 1997
• Reviewed
  26 May 1998