Chemical Composition and Characteristics of Alpha-amylase Inhibitor By A. Y. Gebriel;** A. A. Amin,* M. F. Madkour, ** H. A. El Banna * and K. F. Mahmoud * * Dept. of Food Sci. and Technol., National Research Center, Dokki, Giza, Egypt. ** Dept. of Food Sci., Faculty of Agric. Ain Shams University, Shoubra El-Kheima, Cairo, Egypt. ABSTRACT In this study chemical constituents and characteristics of alpha amylase inhibitor extracted from kidney bean Giza 133 and wheat Giza 164 were studied. Results indicated that the purified alpha amylase inhibitors of kidney bean Giza 133 contained more total carbohydrate content (14.4 %) than wheat Giza 164 (1.2 %), in addition to the differences in amino acid content for both samples. The alpha amylase inhibitor characteristics indicated that the molecular weight of kidney bean Giza 133 (47.000 Da) was higher than both wheat Giza 164 (24.000 Da) and human salivary alpha-amylase (32.000 Da) and lower than enzyme-inhibitor complex (61.000 Da). The low activity of both human and pancreatic alpha amylase was achieved using low concentration of kidney bean inhibitor (1.56 and 1.95 UI) compared with higher concentration (4.69 and 5.47 UI) of wheat Giza 164. The addition of salt (NaCl and bromide) in the purification step increased the inhibitor activity up to 80, 91 %, and the inhibitor reaction rate reached maximum at ratio 1 : 1 (Inhibitor (I) : Enzyme (E)). Results also indicated that the low dissociation constant of kidney bean inhibitor gave stability to the I-E complex at pH 6.9. Increasing the pH values prevent further inhibition and the reaction is irreversible, while raising the temperature up to 37 oC reverse the inhibition reaction to its primary rate. Key Words: Alpha-amylase inhibitor; Wheat; Legumes. INTRODUCTION During the past 10 years considerable progress has been made in the physicochemical properties, nutritional and physiological role of plant alphaamylase and its protein inhibitors. Most of these inhibitors are inactive toward plant alpha-amylases references suggesting their physiological role is not to regulate alpha-amylase levels in the plant, but controlling starch metabolism (Octavio and Rigden, 2002). Purified alpha-amylase inhibitors are glycoproteins and active against human pancreatic, salivary and insect alpha-amylase. They are inactive against bacterial, mold and plant alpha-amylases (Frels and Rupnow, 1984 and Santimone et al. 2004). Alpha amylase inhibitor have similar amino acid compositions, molecular weight (49,000 Daltons) and contained two or more subunits, with molecular weights ranging from 20,000 to 60,000 daltons and emerging similarities in N-terminal amino acid residues and partial amino acid sequences. The amount of inhibitor extracted from whole wheat bread or barley was from 60 to 69 % lower than the corresponding meal. The presence of carbohydrate as apart of the inhibitor molecule has been demonstrated in beans but not in wheat (Saunders and Lang, 1973). The carbohydrate content of alpha-amylase inhibitor and its nature play a specific role in the inhibition mechanism of the enzyme. (Power and Whitaker 1977, Yoshima et al., 1980 and Sharma and Pattabirman, 1982). However, the complete absence of carbohydrate in wheat amylase inhibitors (Silano et al., 1973) and the low carbohydrate content (10 %) of legume inhibitors (Power and Whitaker 1977) decreases the possibility of inhibition process in cereals and legumes. The stoichiometry interaction between the alpha-amylase and wheat inhibitor showed a binding complex of 1:1 enzyme : inhibitor. The purified alpha-amylase inhibitor from wheat or legumes presents great potential for use in phaseulus genetic improvement programs Octavio et al. (2005). All characteristics indicated that the inhibitors act as highly specific substrates for the enzyme, and inhibit at a unique peptide bond called the reactive site peptide bond. Although the reaction inhibition mechanisms are not clearly understood, yet reducing sugars, which are covalently bound to the inhibitor polypeptides chain, may play a major role in it, or the inhibitor may induce conformational changes in the enzyme molecule (Heidari, et al., 2005). Therefore the present work was aimed to study the chemical composition of alpha-amylase inhibitor extracted from wheat grains (Giza 164) and Kidney bean (Giza 133), In addition the inhibitors characteristics and enzyme inhibitor mechanism were also studied. MATERIAL AND METHODS Materials: Alpha amylase inhibitors [extracted from wheat Giza 164 and kidney bean Giza 133] and purified according to Mahmoud, 2006 were used. Human pancreatic and salivary alpha-amylase were obtained from Sigma Company, England. Methods: Chemical composition of the purified alpha-amylase inhibitor: Carbohydrate composition: The carbohydrate content of the amylase inhibitor was determined either in the whole inhibitor by using the phenol-sulfuric acid reaction of Dubois et al. (1956) or after separation of the carbohydrate moiety, as follows: 2 mg of the inhibitor was hydrolyzed with 2 N HCl for 10 h at 100 oC in a closed Pyrex tube under nitrogen. The solution was then passed through 2 ml Bio-Rad AG1-X-2 column, and the free sugars were eluted with 70 % ethanol. To separate neutral sugars from amino sugars, a Bio-Rad AG50W-X2 column was used and sugars obtained were determined according to Krystal and Graham (1976). The identification of individual carbohydrates was accomplished on whatman paper chromatography (1 MM) (Power and Whitaker, 1977). Amino acids analysis of alpha-amylase inhibitor: Amino acids analysis was performed in a Beckman 120C analyzer according to Spackman et al. (1958). After hydrolysis with 6 N HCL in closed tubes, under nitrogen, at 110 C, and for 72 h. Cysteine and methionine were determined respectively as cysteic acid and methionine sulfone by oxidation with performic acid (Moore, 1963). Tryptophan was determined according to Beaven and Holiday (1952). For amino sugar determination, the inhibitor was hydrolyzed with 4 N HCl at 110 oC for 4 and 6 hr (Spiro, 1973). Terminal amino acids were identified basically as described by Gray, (1972). Electrophoresis in polyacrylamide gel rods were run either in alkaline pH according to Davis, (1964) or in acid media as proposed by Reisfield et al. (1963), and in neutral solution by Weber and Osborn (1969). Protein bands were visualized by staining with coomassie brilliant or by the reaction with fucsine to demonstrate glycoproteins. The cross-linking reaction with dimethyl suberimidate was performed as described by Davis and Stark (1970). Dissociation of the inhibitor was studied by using different media: SDS (1%), urea (6M), guanidine (6M), and β-mercaptoethanol (1%) in 0.1 M phosphate buffer, pH 6.9, according to Tanford (1968). The elimination of the reagents after the dissociation was done by dialysis against deionized water for 24 hr followed by dialysis against 0.1M phosphate buffer, pH 6.9; SDS was removed from the media by using an ion-exchange (AGI-X8) resin (Lenard, 1971). Determination of amylase activity: Alpha-amylase activity was determined according to the method described by Bernfeld (1955). Alpha-amylase inhibitor assay: The alpha-amylase inhibitor activity was determined using dinitrosalicylic acid method described from Bernfeld, (1955) and modified by Ishimoto and Chrispeels (1996). One unit of inhibitor activity is defined as the amount which causes approximately 50 % inhibition of α-amylase activity under the assay conditions. 2 Some characteristics of the purified alpha-amylase inhibitor: Molecular weight of alpha-amylase inhibitor: The molecular weight of the inhibitor was estimated by SDS-PAGE using the method of Laemmli (1970). Purified inhibitor, human salivary and pancreatic alpha-amylase activities: The purified alpha amylase inhibitor and its effect on human salivary and pancreatic alpha amylase activities was determined according to Hoover and Sosulski (1984). Alpha-amylase inhibitor activity at different salts: The effect of salts i.e. (NaCl, KCl, Br- and I2 ) on alpha amylase inhibitor incubated for 30 min at 30 oC with sodium phosphate buffer (0.02M, pH 6.9) was determined according to the methods described by O'Donnell and McGeeney (1976). Starch hydrolysis by alpha-amylase in present of its inhibitor: Human pancreatic alpha amylase was determined in three digests according to Ceska et al. (1969). Isoelectric point of alpha-amylase inhibitor: Isoelectric point of the purified inhibitor was determined using polyacrylamide gel electrophoresis (pH 4 to 6) according to the method described previously, by Wrigley (1971). Stoichiometry of inhibition: Amylase inhibitor (4.5 unit inhibitor/mg protein) was incubated with human alpha-amylase (4.1 unit/mg protein) in sodium phosphate buffer (0.02 M, pH 6.9) containing NaCl (0.05 M) at 30 oC. A control experiment containing only alpha-amylase was incubated under identical condition. Aliquots containing 0.6 ml were removed at interval up to 10 h and transferred to tubes containing 0.35 ml of starch as a substrate (8 mg / ml). Alpha-amylase activity was determined according to the usual assay procedure (Jane and John, 1985), and then the procedure was repeated as follows: 1- Inhibitor + substrate. And added enzyme. 2- Inhibitor + enzyme. And added substrate. 3- Substrate + enzyme. And added inhibitor. Dissociation constant: Dissociation constant of alpha amylase and its inhibitor was measured by using the method described by Bieth (1974). Reversibility of inhibition: The effect of high pH and low temperature on alpha amylase inhibitor was determined according to John and Carmen (1975). Specificity of alpha-amylase inhibitor: The purified inhibitor was tested for its ability to inhibit alpha-amylase from a number of sources i.e. plant, animal, fungal, and bacterial according to Franco and Flavio (1985). RESULTS AND DISCUSSION Chemical analysis of alpha-amylase inhibitor from wheat flour Giza 164 and kidney bean Giza 133. 1- Carbohydrate composition: Carbohydrate composition of alpha-amylase inhibitor for both wheat flour and kidney bean is shown in table (1). 3 Table (1): Carbohydrate composition of alpha-amylase inhibitor from both wheat flour and kidney bean. Sources of alphaamylase inhibitor Total carbohydrate % Sugars % after hydrolysis by phenolsulfuric acid reagent (H2SO4 0.1 N) Neutral sugar % Amino sugar % Mannose 0.3 ---1.2 Galactose 0.9 ---Mannose 1 ---Kidney bean Xylose 2.4 ---14.4 Giza 133 Galactose 8.0 ----------Glucosamine 3 Results in table (1) showed that the glycoprotein nature of the purified kidney bean Giza 133 inhibitor contained more total carbohydrate content than that in wheat flour Giza 164 inhibitor being 14.4 and 1.2 % respectively, and that it contained about 11.4 % neutral sugar (i.e. mannose, xylose and galactose) with 3 % amino sugar i.e. (glucosamine), while wheat flour inhibitor contained only 1.2 % neutral sugar mainly mannose and galactose and with no amino sugar content. Wheat flour Giza 164 2- Amino acid analysis of alpha-amylase inhibitor: Amino acid composition of the wheat flour Giza 164 and kidney bean Giza 133 are shown in table (2). Values are given as mol of residue per mol of protein after 24 hr hydrolysis in 6 N HCl at 100 oC. Table (2): Amino acid analysis of alpha-amylase inhibitor from both wheat flour and kidney bean (per mol of protein). Amino acid Asparagines Thereonine Serine Glutamine Proline Glycine Alanine Cysteine Half-Cystine Valine Methionine Inhibitor Analysis (mol %) Wheat Kidney flour bean Giza 164 Giza 133 15.5 77.1 5.7 32.4 14.7 52.2 23.6 36.1 15.3 13.5 22.4 20.3 29.1 23.6 16.8 1 8.8 --19.1 36.0 4.6 2.3 Amino acid Iso-Leucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Trptophane Glucosamine C.M.Cysteine Inhibitor Analysis (mol %) Wheat Kidney flour bean Giza 164 Giza 133 4.9 18.2 19.4 20.1 9.1 16.4 3.8 23.1 2.2 15.0 7.1 2.0 12.0 13.3 3.1 2.0 --2.1 18.5 --- Results in table (2) showed that there were differences in amino acid content for both wheat flour Giza 164 and kidney bean Giza 133 inhibitors. Kidney bean had higher contents of asparatic acid, serine, glutamic acid and thereonine being 77.1, 52.2, 36.1 and 32.4 mol %, respectively. Wheat flour had higher content of Alanine (29.1 %), glutamic acid (23.6 %) and glycine (22.4 %). Results also indicated that wheat flour Giza 164 inhibitor had 8.8 % half-cystine which is slightly less than that found in malted barley inhibitor being 9 % (Randall et al., 1983). The amino acids liberated with time as C-terminal i.e. leucine and tyrosine and third C-terminal amino acid i.e. serine, alanine and asparatic acid is given in figure (1). 4 Moles Librated 25 20 15 10 5 0 0 10 20 30 40 50 60 70 80 90 100 Min Leu Tyr Ala Ser Asp Val Figure (1): Identification of liberated amino acid. 2- Characterization of the purified alpha-amylase inhibitor Molecular weight estimation of alpha-amylase inhibitor Molecular weight of the two purified wheat flour Giza 164 and kidney bean Giza 133 inhibitors were estimated and the results are shown in figures (2 and 3). Ribo-Nuclease 0.5 Alpha-Chymo Trypsinogen 0.4 Inhibitor 24.000 Da Bovine Serum Albumin 4. 5 4. 4 4. 3 4. 2 0 4 Ov-Albumin 5 0.1 Beta-Lactoglobulin 4. 9 0.2 Complex 41.000 Da Alpha-amylase 32.000 Da 4. 8 0.3 4. 7 0.6 4. 6 Kda (Kilo Dalton) 0.7 Fraction num ber Com plex Alpha-am ylase Inhibitor Figure (2): Molecular weight of purified wheat flour inhibitor, human salivary alpha-amylase and alpha-amylase-inhibitor complex. Results in figure (2) indicated that the molecular weight of wheat flour Giza 164 inhibitor was approximately 24,000 Da, which is in agreement with that found by Randall et al. (1983) suggesting that the inhibitor is not a dimer. Results also showed that the molecular weight of human salivary alpha-amylase was 32,000 Da which is low compared to the value reported by Greenwood, (1968) being 45,000 Da. Results also showed that gel filtration of the inhibitor with human salivary alphaamylase at pH 8.0 yielded a new peak having a molecular weight of 41,000 Da, presumably, due to the formation of enzyme-inhibitor complex. These results are in agreement with that demonstrated by Weselake et al. (1983). 5 RiboNuclease 0.6 0.5 Alpha-Chymo Trypsinogen 0.4 Complex 61.0000 Da 0.3 Alpha-amylase 32.000 Da Inhibitor 47.000 Da 0.2 Beta-Lactoglobulin 5 4. 9 4. 8 4. 5 4. 4 4. 3 4 4. 2 0 4. 7 Ov-Albumin Bovine Serum Albumin 0.1 4. 6 Kda (Kilo Dalton) 0.7 Fraction num ber Com plex Alpha-am ylase Inhibitor Figure (3): Molecular weight of purified kidney bean inhibitor, human salivary alpha-amylase and alpha-amylase-inhibitor complex. Results in figure (3) indicated that the molecular weight of the purified kidney bean Giza 133 inhibitor, human salivary alpha-amylase and enzyme-inhibitor complex was 47,000, 32,000 and 61,000 Da, respectively. Alpha-amylase Activity % Effect of the purified inhibitor on human salivary and pancreatic alphaamylase activity. The effect of the purified wheat Giza 164 inhibitor on human salivary (HSA) and pancreatic alpha-amylase (HPA) is shown in figure (4). The results indicated that the relative ratio of inhibition was 1.0 : 0.5, and its magnitude was linear against inhibitor concentration. Beyond this point the inhibition was progressive but non linear, reaching nearly 100 % inhibition at high concentrations. No activity of both human salivary and pancreatic alpha-amylase was achieved when inhibitor concentration was 6 and 7 μg of protein, respectively. 100 90 80 70 60 50 40 30 20 10 0 0 1 2 3 HSA 4 5 6 7 8 9 10 Inhibitor Concentration (μg) HPA Figure (4): The purified wheat Giza 164 inhibitor and the activity of HSA & HPA. HSA: Human Salivary Amylase -- HPA: Human pancreatic Amylase HSA 10 9 8 7 6 5 4 3 2 1 100 90 80 70 60 50 40 30 20 10 0 0 Alpha-amylase Activity % The same experiment was repeated using the purified kidney bean Giza 133 inhibitor on both enzymes figure (5). Inhibitor Concentration (μg) HPA Figure (5): The purified kidney bean Giza 133 inhibitor and the activity of HSA & HPA. 6 The results showed that the low activity of both human salivary and pancreatic alpha-amylase was achieved when inhibitor concentration was 2.0 and 2.5 μg of protein, respectively. So, the purified inhibitor from kidney bean Giza 133 was chosen to study its characteristics and properties because: Firstly, the amount of the inhibitor used is small and capable to inhibit the activity of both human salivary and pancreatic alpha-amylase comparing with the amount used from the purified wheat Giza 164. Secondly, wheat flour Giza 164 inhibitor was not heat stable and heat treatment at 50 and 70 oC for 10 min caused 50 and 100 % loss in its activity, respectively. While, storage at 4 oC for 2 weeks caused also 50 % loss in activity, and also below pH 4.0 the loss reached 60 %. The inhibitor was maximally stable at pH 6.9 (Mahmoud, 2006). 100 90 80 70 60 50 40 30 20 10 0 HSAI HPAI % Inhibition c 50 40 30 20 10 0 100 90 HPAI d 80 70 60 50 40 30 20 Br¯ (mg) HSAI 0.9 0.7 0.5 0.1 0.9 0.7 0.5 0.3 0.1 10 0 0.3 % Inhibition 100 90 80 70 60 0.9 KCl (mg) NaCl (mg) HSAI 0.7 0.5 b 0.1 0.9 0.7 0.5 0.3 a 0.3 % Inhibition 100 90 80 70 60 50 40 30 20 10 0 0.1 % Inhibition Effect of salts on alpha-amylase inhibitor activity: It was found that the purified alpha-amylase inhibitor lost its activity after dialysis by the early purification steps. Such activity could be restored by adding small amounts of NaCl to the media. The ionic strength of the pre-incubation buffer had a profound effect on the inhibitory activities of alpha-amylase inhibitor (figure 6). I¯ (mg) HPAI HSAI HPAI Figure (6): Salts ionic strength, inhibitor of salivary and pancreatic alpha-amylase activity. Results in figure (6) showed that the activities of inhibitor at 0.9 ionic strength were 25 % more than 0.1 and were in agreement with the activities reported by Jane and John (1985). The same results were also observed with Bromide. These results suggested that an increase in salt ionic strength might facilitate inhibition by shielding charge groups on the enzyme or inhibitor, which could cause a conformational change or an alkaline shift at the optimum pH. Although 7 O`Donnell and McGreeney (1976) reported that increasing phosphate buffer concentrations from (0.1 to 50 mM) did not have any effect on the degree of inhibition of salivary alpha-amylase by inhibitor isolated from wheat, while the addition of NaCl (1 mg) to phosphate buffer (0.1 mM), increased the inhibitor activity from 68 to 85 %. The results also showed that addition of KCl and iodine up to 1.0 mg have no effect on the inhibition of human salivary and pancreatic alpha-amylase by the inhibitor. iodine stain (% of original value) Starch hydrolysis by alpha amylase in presence of its inhibitor: The effect of starch on the interaction between kidney bean Giza 133 inhibitor and human pancreatic alpha-amylase was determined. Figure (7) showed that whether inhibitor can interact with alpha-amylase in the presence of substrate (starch), or substrate can dissociate the enzyme-inhibitor complex. Results in figure (7) showed that there is a noticeable decrease in iodine staining power (digest b) after 10min incubation indicating that starch can interact with inhibitor–enzyme complex causing reduction in its amount. 120 100 a 80 60 b 40 c 20 0 0 10 20 30 40 50 60 Time of Incubation (min) Figure (7): Degradation of starch by human pancreatic alpha-amylase. Digest (a): starch solution added after incubation at 37 oC for 30 min. Digest (b): addition of starch to enzyme-inhibitor mixture. Digest (c): Addition of inhibitor to enzyme-substrate mixture. Above results showed that either formation of Enzyme-inhibitor or enzymesubstrate and addition of inhibitor caused a reduction in the hydrolysis action of enzyme. Such effect could be attributed to the formation of enzyme-inhibitor-substrate and enzyme-substrate-inhibitor by the some rate and gave the same results. Isoelectric point of alpha-amylase inhibitor: The isoelectric point of kidney bean alpha-amylase inhibitor obtained by using polyacrylamide gel electrophoresis is shown in figure (8). Solubility % 100 80 60 40 20 0 3.5 4 4.5 5 5.5 6 pH value Figure (8): Isoelectric point of alpha-amylase inhibitor Results showed that the isoelectric point of kidney bean alpha-amylase inhibitor was at 4.5, similar to that obtained by Power and Whitaker (1977) being 4.65. The inhibitor isolated from wheat had higher isoelectric points (around 6.7) and curiously also had higher pH optimum, reaching values in the alkaline range which is in agreement with that reported by Saunders and Lang (1973). 8 Stoichiometry of inhibition: The stoichiometry of inhibition alpha-amylase interaction was investigated during a different period of pre-incubation i.e. 0 to 60 min, and the maximum extent of inhibition was given in figure (9). INHIBITION (%) 100 80 60 40 20 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Duration of Preincubation (m in) Figure (9): The determination of the stoichiometry of inhibitor-human salivary alpha-amylase. Results showed that the maximum extent of inhibition 93 % was observed when the 0.40 μg of inhibitor (MW 47.000 daltons) and at 60 min pre-incubation was added to 0.34 μg alpha-amylase (MW 32.000 daltons), suggesting that the inhibition reaction was approximately at ratio 1 : 1. These results are in agreement with that reported by John and Carmen (1975) and Makoto et al. (1987). Dissociation constant: The dissociation constant for kidney bean alpha-amylase inhibitor against pancreatic alpha-amylase is shown in figure (10). 60 (1-a) (10-9) I0 ----------------------- 50 40 30 20 10 0 0 2 4 6 8 10 12 14 1 ----a 16 Figure (10): Dissociation constant of the complex of human pancreatic alpha-amylase and the inhibitor from kidney bean. Where: (I) the initial inhibitor concentration (a) the activity of free enzyme without inhibitor Results in figure (10) showed that the dissociation constant of complex kidney bean Giza 133 inhibitor with pancreatic alpha-amylase was 2.4 X 10-9 M, higher from that reported by Power and Whitaker (1977) being 3.5 X 10-11 M for red kidney bean indicating that the lower the dissociation constant is the stable the inhibitor-enzyme complex forming is at pH 6.9. Reversibility of inhibition: Attempts were made to determine whether the conditions found to be unfavorable for inhibition, namely high pH and low temperature, will reverse inhibition, which has already taken place. Effect of pH The activities of alpha-amylase comparing with those in appropriate control digests from which the alpha-amylase inhibitor was omitted. Results indicated that pre-incubation at pH 5.5 followed by pre-incubation at pH 8.2 gave alpha-amylase activity identical to each other, but lower than that after pre-incubation at pH 6.9. Thus, elevation the pH prevents further inhibition, and doesn't reverse inhibition, which has already occurred. 9 Inhibition % Effect of low temperature The effect of low temperature on alpha-amylase inhibitor activity at pH 6.9 and after pre-incubation of an identical digest under the same conditions for the same length of time, followed by 45 min at 0 oC, is shown in figure (11). 100 80 60 40 20 0 0 15 30 45 60 75 90 Time (min) Reversible Figure (11): Effect of low temperature at (0 oC) on reversibility of human salivary alpha-amylase inhibition. Results showed that at low temperature (0oC) the inhibition reduced with time up to 45 min (being 51 %), followed by stability stage up to 90 min. Raising the temperature up to 37oC reversed the inhibition reaction to its primary rate. Specificity of kidney bean alpha-amylase inhibitor The purified kidney bean inhibitor was tested for its ability to inhibit alpha amylase from different sources i.e. plant, animal, fungal and bacteria, and the results are given in table (3). Table (3): Specificity of kidney bean Giza 133 alpha-amylase inhibitor against different sources of alpha-amylase. Sources of alpha-amylase Extent of inhibition % Animal 95 Human salivary 100 Human pancreatic 97 Hog pancreatic Bacteria 5 Bacillus subtilis 0 Bacillus licheniformis Fungus 3 Aspergillus oryzae Plant 0 Wheat 0 Kidney bean 0 Barley Results in table (3) showed a complete inhibition of human pancreatic alphaamylase and reduction to 95 and 97 % for human salivary and hog pancreatic alpha-amylase, respectively. The inhibitor was also active on bacteria and fungus i.e. Bacillus subtilis (5 %) and Aspergillus oryzae (3 %) but not on plant i.e. Wheat, Kidney bean and Barley. These results are in agreement with that reported by John and Carmen (1975), Fronco and Flavio (1985) and Ramanaoppa and Thillaisthanam (1985). Conclusion: It could be concluded that kidney bean inhibitor reaction rate reached maximum at 93 % when 0.4 µg inhibitor at 60 min pre-incubation was added to 0.34 µg alpha-amylase indicating that the inhibitor reaction was at ratio 1 :1. REFERENCES Beaven G. H. and Holiday E. R. (1952). Evaluated of tryptophan in cereal and legume. Adv. Protein Chem., 7, 319-324. Bernfeld P. (1955). Amylases, α and β. In "Methods in Enzymology" Vol. 1: 149-154. 10 Bieth J. (1974). Some kinetic consequences of the tight binding of protein-proteinase-inhibitors to proteolytic enzymes and their application to the determination of dissociation constants. In "Proteinase Inhibitors," Bayer Symposium V, Ed.) H. Fritz, H. Tschesche, L. J. Greene, and E. Truscheit. Springer-Verlag, New York. Ceska M., Birath K. and Brown B. (1969). A new and rapid method for the clinical determination of alpha-amylase activities in human serum and urine. Optimal conditions. Clin, Chem. Acta, 26, 437-446. Davis G. E. and Stark G. R. (1970). Protein bands in alpha-amylase inhibitor and cross-linking reaction with dimethyl suberimidate. Natl. Acad. Sci. U.S.A., 66, 651-657. Davis B. J. (1964). Disc-electrophoresis II-method and application to human serum proteins. Ann. N. y. Acad. Sci. 121, 404-427. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A. and Smith F. (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356. Franco M. L. and Flavio F. F. (1985). Partial characterization of the amylase inhibitor of black beans (Phaseolus vulgaris), variety Rico 23. J. Agric. Food Chem., 33, 132-138. Frels J. M. and Rupnow J. H. (1984). Purification and partial characterization of two alpha amylase inhibitors from black bean (Phaseolus vulgaris). J. Food Biochem. 8: 281-290. Gray W. R. (1972). Terminal amino acids were identified from alpha-amylase inhibitor. Methods Enzymol., 25, 121-124. Greenwood C. T. (1968). Starch degrading and synthesizing enzymes: A discussion of their properties and action patterns. Adv Carbohydrate Chem., 23, 281-366. Heidari R., Zareae S. and Heidarizadeh M. (2005). Extraction, Purification, and inhibitory effect of alpha-amyase inhibitor from wheat (Triticum aestivum Var. Zarrin). Pakistan Journal of Nutrition, 4, 101-105. Hoover R. and Sosulski F. S. (1984). Characteristics and concentrations of alpha-amylase inhibitor in phaseolus vulgaris biotypes, starch/stärke 36, (7): 246-250. Ishimoto M. and Chrispeels M. J. (1996). Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean. Plant Physiol., 111, 393-401. Jane M. F. and John H. R. (1985). Characterization of two alpha-amylase inhibitors from back bean (Phaseolus vulgaris). Journal of Food Science, Vol. 50, No. 1, pp. 72-77. John J. M. and Carmen M. L. (1975). Purification and properties of phaseolamin, an inhibitor of alpha-amylase, from the kidney bean, Phaseolus vulgaris. The Journal of Biological Chemistry, Vol. 250, No. 20, pp. 8030-8037. Krystal G. and Grahan A. F. (1976). A sensitive method for estimating the carbohydrate content of glycoproteins. Anal. Biochem. 70, 336-345. Laemmli U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Mahmoud F. K. (2006). Studies on Alpha amylase inhibitors in cereals and legumes. MSC, Thesis, Faculty of Agriculture Ain Shams University. Makoto K., Hideki Y., Tsuneo I., Koichi S., Kimikazu I. and Fumio I. (1987). An alpha-amylase inhibitor from cranberry bean (Phaseolus vulgaris): its specificity in inhibition of mammalian pancreatic alpha-amylases and formation of a complex with the porcine enzyme. J. Nutr. Sci. Vitaminol., 33, 359-367. Moore S. (1963). Determine of cysteine and methionine as cysteic acid and methionine sulfone by oxidation. J. Biol. Chem. 238, 235-237. Oُ Donnell M. D. and McGeeney K. F. (1976). Purification and properties of an alpha-amylase inhibitor from wheat. Biochem. Biophys. Acta.422, 159-169. Octavio, L. F. and Rigden D. (2002). Plant alpha-amylase inhibitors and their interaction with alpha-amylase. Eur. J. Biochem., 269: 397-412. Octavio L. F., Francislete R. M., Paulo A. M., Norma S. P., Massaru Y., Marise V. C., Carlos B. J. and Maria F. G. (2005). Characterization of two Acanthoscelides obtectus alpha-amylases and their inactivation by wheat inhibitors. J. Agric. Food Chem., 53, 1585-1590. Power J. R. and Whitaker J. R. (1977). Effect of several experimental parameters on combination of red kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor with porcine pancreatic alphaamylase. J. Food Biochem., 1, 239-260. Ramanakoppa H. N. and Thillaisthanam N. P. (1985). Isolation of an amylase inhibitor from Setaria italica grains by affinity chromatography on Blue-Sepharose and its characterization. J. Agric. Food Chem., 33, 645-650. Randall J. W., Alexander W. M., Robert D. H. and Harry W. D. (1983). Purification and characterisitic of an Endogenous alpha-amylase inhibitor from wheat kernels. Plant Physiol., 73, 1008-1012. Reisfield R. A., Lewis V. I. And Williams D. E. (1963). Electrophoresis of amino acid from alphaamylase inhibitor in polyacrylamide gel rods in acid media, Nuture. (London), 196, 281-285. Santimone M., Koukiekolo R., Moreau Y., Le-Berre Y., Rouge P., Marchis-Mouren G., and Desseaux V., (2004). Porcine pancreatic alpha amylase inhibition by the kidney bean (Phaseolus vulgaris) inhibitor and structural changes in the alpha amylase inhibitor complex. Biochim. Biophys. Acta, 1696, 181-190. Saunders, R. M. and Lang J. A. (1973). Alpha-amylase inhibitors in Triticum aestivum: purification and physical-chemical properties. Phytochem., 12: 1237-1246. Sharma K. K. and Pattabirman T. N. (1982). Natural plant enzyme inhibitors. Purification and properties of an amylase inhibitor from yam (Dioscorea alata). J. Sci. Food Agric., 33, 255-262. Silano V., Pocchajri F. and Kasarda D. D. (1973). Physical characterization of six alpha-amylase inhibitors from wheat. Biochem. Biophys. Acta, 317, 139-146. Spackman D. H., Stein W. H. and Moore S. (1958). Performed amino acid analysis in Beakman 120 analyzer. Anal. Chem., 30, 1190-1196. Spiro R. G. (1973). Determined amino sugar from inhibitor and hydrolyzed with hydrochloric acid. Adv. Protein Chem. 27, 349-352. Tanford, C. (1968). Dissociation of alpha-amylase inhibitor by using different media. Adv. Protein Chem., 23, 121-125. 11 Weber K. and Osborn M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. of Biol. Chem., 244 (16), 4406-4412. Weselake R. J., MacGregor A. W., Hill R. D. and Duckworth H. W. (1983). Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels. Plant Physial., 73, 1, 8-13. Wrigley C. M. (1971). Estimate of isoelectric point of the alpha-amylase inhibitor from black beans. Methods Enzymol., 22, 559-562. Yoshima H., Furthmays H. and Kobata H. (1980). Structural features of red kidney bean alphaamylase inhibitor in binding with alpha-amylase. J. Biol. Chem., 255, 9713-9719. 12