J. Sci. Fd Agric. 1916, 27, 449-453

Continuous Automated Assay of a-Amylase Inhibitors Luciano Vittozzi, Gino Morisi and Vittorio Silano Labovatori di Chimica Biologica, Istituto Superiore di Sanitd, Roma, Italy (Manuscript received 9 May 1975)

A Technicon AutoAnalyzer was adapted to carry out a continuous assay of a-amylase activity to detect the presence of a-amylase inhibitors. The manifold design is based on the same sequence as the Bernfeld reducing-sugar procedure and allows sufficient time to detect those inhibitors which act slowly. The technique can be used to assay protein amylase inhibitors from wheat at concentrations as low as 1.7 nM. It may also be used to detect amylase activity which can continue in spite of the presence of inhibitors. 1. Introduction

Alpha-amylase inhibitors are widely distributed in the plant kingdoml-6 and occur in the albumin fraction of the wheat kerne1.7-9 Several such inhibitors from wheat are active against insect, avian, and mammalian a-amylases and have been purified and characterised in a number of laboratories.10-15 An a-amylase inhibitor from kidney bean has been isolated and characterised as well.lO, 1 7 Wheat inhibitors act not only in vitro, but also in vivo,18, l 9and have been used as markers to establish phylogenetic relationships in Triticum,8 and to prepare an affinity column for the purification of inhibited amylases.z0 A number of qualitative screening techniques for the analysis of amylase inhibitors including that described by Fossum and Whitaker21 are available, but quantitative assays of amylase inhibitors are time consuming and not readily reproducible. Various automated methods of differing complexity have been described for the amylase analysis, including saccharogenic22 and iodometric23.24 ones. A fluorimetric procedurez5has been described for the analysis of tissue amylase output. The iodine-staining methods cannot be used for a wide range of starch concentration22 and may be affected by inactive proteins if they are present.23 On the other hand, the ferricyanide reducing sugar procedure is very sensitive to interfering reducing material and can be applied only to highly purified amylases.22 Here we report a Technicon AutoAnalyzer system which can carry out a continuous analysis of amylase activity in the presence of its inhibitors. The technique is based upon the Bernfeld reducing sugar procedure26 and can detect inhibitors which act s10wly.l~ 2. Experimental

Standard Technicon (Ardsley, NY, USA) equipment was used consisting of a proportioning pump, two baths (37°C and 95"C),a colorimeter, a 15 mm tubular flow cell, a 525 nm filter, and a recorder. The manifold flow diagram for the amylase inhibition assay (Figure 1) shows the buffer stream (0.3 ml/min), which after segmentation, met the sample effluent and amylase streams (0.02 ml/min and 0.19 mllmin, respectively). The resulting mixed stream was passed to the first time-delay coil which allowed a 30 min interaction between the amylase and the inhibitor. Then the starch stream (0.45 ml/min) was mixed. The incubation took place in a 5 min delay coil. Both the pre-incubation and the incubation coils were kept in a water bath at 37°C. The DNSA reagent stream (0.6 ml/min) stopped the enzymatic reaction and a 20 min heating period in the 95°C bath allowed the develop449

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Flow cell

DNSA

Starch Air

Buffer Column effluent Amylase

37°C Incubation bath

Figure 1. Flow diagram for the continuous automated assay of cc-amylase inhibitory activity. Technicon equipment was used in the construction of the analytical system.

ment of the red colour. After cooling the reaction mixture was passed through a microdebubbler before entering the colorimeter for absorbance measurement. The calibration of the AutoAnalyzer system was checked by measuring the absorbance given by maltose solutions of known concentrations varying in the reaction mixture between 0.022 and 0.264 mM. The maltose solutions entered the amylase tube and the column effluent tube was filled with buffer. The buffer solution contained 0.15 M-NaCl, 0.02 M-sodium acetate, 0.02 M-sodium barbiturate and 0,001 M-CaClz; the buffer pH was adjusted with 1 M-HCl to 6.9 for human saliva amylase and to 5.4 for Tenebrio molitor L. amylase. The human saliva cc-amylase used was freeze-dried crude saliva, and T. malitor L. cc-amylase was prepared according to Applebaum et al.27 Hog pancreas cc-amylase was supplied by Merck (Darmstadt, West Germany). The amylase stock solution was prepared by dissolving the enzyme in the appropriate buffer at a concentration of about 70 a.u./ml. One amylase unit (a.u.) is the amount of enzyme that produces, under our experimental conditions, 1 pequivalent of maltose per min. The solution was stored at -20°C and diluted just before use. The starch suspension was prepared by gelatinising 5 g soluble starch (Carlo Erba, Milan, Italy) in 100 ml of boiling water, and then diluting up to 1 litre with buffer. The dinitrosalicylate (DNSA) reagent was prepared by dissolving 5 g dinitrosalicylic acid (Merck, Darmstadt, West Germany) in 200 m12 M-NaOH. After dilution with 250 ml distilled water, 150 g potassium sodium tartrate was added. The mixture was further diluted with water to 750 ml. The starch, buffer and DNSA reagent solutions were filtered through a sintered glass and wetting agent ARW-7 was added up to 0.15 % (v/v) before use. A highly purified albumin characterised by Silano et ul.13928 (coded 0.19) was obtained from the wheat kernel according to the procedure of Sodini et al.12 The water extract from the wheat kernel (Triticum aestivum var. Chinese spring) was subjected to gel filtration on a 2 x 110 cm column of Sephadex G-100 after the procedure described by Bedetti et

3. Results and discussion

A direct recording taken from the manifold [Figure 2(a)] showed that a maltose concentration as low as 0 . 0 2 2 m ~could be accurately detected. A linear response was obtained throughout the

Assay of amylase inhibitors

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W-IA Figure 2. (a) Recorder trace for the maltose standard solutions. Maltose solutions were assayed with the dinitrosalicylate reagent at a sampling rate of three samples per hour. A 5 min washing was carried out after each assay. (b) Plot of the peak height o n the recorder trace versus the concentration of maltose. (c) Reproducibility of maltose determinations.

maltose concentration range tested [Figure 2(b)] and the standard deviation derived from four analyses was less than 2 % of the actual absorbance [Figure 2(c)]. An identical linear pattern was obtained when testing, with soluble starch as substrate, human saliva and T. molitor or-amylases at concentrations between 0.01 and 0.09 a.u./ml in the assay mixture. In this case the column effluent tube was filled with buffer. Although the system as described has been set up to assay or-amylase activity, the manifold may detect @-amylaseactivity as well. However, no calibration experiments for @-amylaseassay have been carried out. An amylase concentration of 0.05 a.u./ml was chosen for amylase inhibition assays with the 0.19 albumin inhibitor from the wheat kernel12913 at the concentrations indicated in Figure 3(a). By plotting the per cent inhibition or absorbance decrease versus inhibitor concentration, we obtained a linear slope up to 40 % inhibition of the amylase present [Figure 3(b)]. This inhibition pattern agreed with those obtained by other authors’, 14328 who tested different protein inhibitors from wheat kernel with manual techniques. The good reproducibility of the assay was checked by repeating the analyses three times at two different inhibitor concentrations [Figure 3(c)]. For these discontinuous assays a minimum volume of inhibitor solution of 0.3 ml is required. A Sephadex G-100 column loaded with a water extract from the wheat kernel was used for the continuous assay of human saliva and T. molitor amylase inhibitors. Two similar manifolds (one for the mammalian and the other for the insect amylase) were used simultaneously. According to previous results with manual techniques, 8 the automated continuous analysis showed the presence in this extract of three inhibitor peaks with different apparent molecular weights active toward the

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Figure 4. (a) Automated continuous-flow assay of or-amylase inhibitors in the effluent of a Sephadex G-100 column loaded with a water extract from the wheat kernel. Human saliva amylase (dashed line); T. niolitor L. amylase (solid line). (b) Automated continuous-flow assay of human saliva amylase inhibition (dashed line) and of amylase activity (dotted line) in the effluent of a Sephadex G-100 column loaded with the water extract of Figure 4(a) added with hog pancreas or-amylase.

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insect amylase [Figure 4(a)]. The two peaks with higher molecular weights were also active toward human saliva amylase. The two-manifold AutoAnalyzer system permitted an accurate comparison to be made of the elution volumes of peaks of the inhibitor active against the two amylases. Figure 4(a) also shows the constancy of the amylase and starch baselines throughout the whole analysis which lasted about 15 h. When one manifold was used to assay a-amylase inhibition and the other to determine amylase activity in the column effluent, the two manifold AutoAnalyzer system also made possible the detection of non-inhibited cr- or P-amylase activity which was possibly interfering with the assay of cr-amylase inhibitors. This is illustrated in Figure 4(b) which compares the gel filtration inhibition (dashed line) and amylase (dotted line) patterns of the wheat extract of Figure 4(a) added to eight units of a hog pancreas amylase very poorly inhibited by wheat albumin inhibitors. 7, lo*l 5 The presence of non-inhibited amylase activity together with protein inhibitors active toward exogenous amylase is not unusual in plant seed extracts.l5 Since different a-amylases liberate oligosaccharides with different degrees of polymeri~ation~~, which may not give an identical reaction with the DNSA reagent,31,32 this method cannot be used for absolute amylase assay, unless the amylase source is known and a calibration curve with the purified amylase is available. References 1.

2. 3. 4. 5. 6. 7.

a.

9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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