PURIFICATION AND PARTIAL CHARACTERIZATION OF WHITE KIDNEY BEAN (PHASEOLUS VULGARIS)β-AMYLASE INHIBITORS FROM TWO EXPERIMENTAL CULTIVARS

β-Amylase inhibitors WKB 858B and WKB 858B were purified to homogeneity from different cultivars of white kidney beans by extraction from the ground beans and by sequential heat treatment, ethanol fractionation, DEAE-cellulose chromatography, Sephadex G-75 gel chromatography and CM-cellulose chromatography. The inhibitors were homogeneous by 7.5% polyacrylamide gel electrophoresis; no isoinhibitors were found. Inhibitors WKB 858A and WKB 858B had isoelectric points of 5.0 and 4.65, respectively, and molecular weights of 42,000 and 20,000, respectively, by FPLC Superose 12 gel filtration chromatography. Inhibitor WKB 858A had molecular weights of 40,000 and 38,000 by Sephadex G-75 gel filtration chromatography and by native gel electrophoresis, respectively. Inhibitor WKB 858A contained 11.0% carbohydrate, N-linked to asparagine residues, with a composition of 1 fucose, 1 xylose, 4 galactose, 8 N-acetylglycosamine and 13 mannose residues per mol of inhibitor. Amino acid analysis of Inhibitor WKB 858A gave a high content of Asx, Glx, Ser, Thr and Val (combined total of 60% molar ratio) and low content of sulfur amino acids (0.8% molar ratio of Met and no 1/2 cystine). No-SH groups were found. The amino acid composition was similar to that of eight other a-amylase inhibitors from beans. Inhibitor WKB 858A formed a 1:1 stoichiometric complex with porcine pancreatic a-amylase with a Ki of 1.0 × 10^?11 M at pH 5.4 and 30C; it had no trypsin inhibitory activity. At pH 6.90 and 30C, the rate of complex formation between Inhibitor WKB 858A and porcine pancreatic β-amylase was 2.76 times faster at 1.385 vs 0.035 ionic strength (with Na₂SO₄), indicating hydrophobic bonds are most important in complex formation.     

PURIFICATION AND SOME PHYSICAL AND CHEMICAL PROPERTIES OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITOR

Red kidney bean contains more amylase inhibitor than do California white bean and cowpea while garbanzo bean and Westan and Westley lima beans do not contain inhibitor. Red kidney bean amylase inhibitor was purified to homogeneity by selective heat treatment (60°C) of a water extract at pH 4. 0, fractionation with ethanol and successive chromatography on DEAE- and CM-cellulose chromatography. The inhibitor has an apparent molecular weight of 49,000 by Sephadex gel filtration and contains 8. 6% carbohydrate probably covalently linked via an amide linkage to asparagine. The inhibitor probably contains four subunits perhaps of three different types. The inhibitor is high in aspartic acid, glutamic acid, serine, threonine and valine, low in cysteine/cystine and does not contain proline. Stable 1:1 complex formation between inhibitor and porcine pancreatic α-amylase was demonstrated by gel filtration on Sephadex G-100. The inhibitor has activity against porcine pancreatic α-amylase, human salivary α-amylase, and Tenebrio molitor (yellow corn meal worm) larval midgut α-amylase but is inactive against Bacillus subtilis α-amylase, Aspergillus oryzae α-amylase, barley α-amylase and red kidney bean α-amylase.     

EFFECT OF SEVERAL EXPERIMENTAL PARAMETERS ON COMBINATION OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITOR WITH PORCINE PANCREATIC α-AMYLASE

Purified red kidney bean (Phaseolus vulgaris) amylase inhibitor forms a 1:1 stoichiometric complex with porcine pancreatic α-amylase leading to complete loss of enzyme activity on starch. Rate of complex formation is pH dependent and is maximal at pH 5. The rate constants for complex formation, as measured by loss of amylase activity, were 2.85 × 10⁴ M^-1 sec^-1 at pH 6.9 (ionic strength of 0.918) and 2.55 × 10⁵ M^-1 sec^-1 at pH 5 at 30°C. At pH 6.9, rate of complex formation was 4.8 times faster at 0.918 ionic strength as compared with the rate at 0.138 ionic strength. At 30°C, pH 6.9 and ionic strength of 0.168 the dissociation constant of the enzyme-inhibitor complex was determined to be 3.5 × 10^-11 M. The rate constant for dissociation of the complex was calculated to be 8.7 × 10^-8 sec^-1 under the same conditions. The rate constant for complex formation, at ionic strength of 0.168, was 1.1 × 10⁴ M^-1 sec^-1 at 37⁰ and 9.77 × 10² M^-1 sec^-1 at 25.7°C. The calculated activation energy for complex formation is 39.5 kcal/mole suggesting a rate-controlling conformational change. Oxidation of the carbohydrate moiety of the glycoprotein inhibitor caused complete loss of activity. Maltose, a competitive inhibitor of α-amylase, bound as readily to the enzyme-inhibitor complex as to free α-amylase. Trypsinized α-amylase, although still able to bind to Sephadex, did not bind inhibitor. The experiments with maltose and trypsinized amylase suggest the inhibitor may not bind at the active site of α-amylase.     

KINETICS OF THE INTERACTION OF PANCREATIC α-AMYLASE WITH A KIDNEY BEAN (PHASEOLUS VULGARIS) - AMYLASE INHIBITOR

An amylase inhibitor isolated from black beans (Phaseolus vulgaris) can completely inhibit porcine pancreatic α-amylase forming a 1:1 stoichiometric complex. The kinetic pattern of complex formation is pH dependent. At pH 5.5 it follows a first order reaction with rate constant of 0.029 min^?1 and 0.017 min^?1 at 37°C and equimolar inhibitor and enzyme concentration, respectively, of 10^?8 M and 10^?9 M. At pH 6.9 it is a second order reaction, with a rate constant of 0.25 × 10⁶ M^?1 min^?1 at 37°C, with 4 × 10^?8 M concentrations of enzyme and inhibitor. The dissociation constants of the enzymeinhibitor complex are 1.7 × 10^?10 M at pH 5.5 and 4.4 × 10^?9 M at pH 6.9, at 37°C. The kinetic data obtained at pH 5.5 suggested the formation of an initial reversible complex followed by a conformational change step. The complex can be dissociated either in acid pH (4.3) or at pH values higher than 6, 5 with partial recovery of the amylase activity.     

COMBINATION OF BLACK BEAN (PHASEOLUS VULGARIS) AMYLASE INHIBITOR WITH MODIFIED PANCREATIC α-AMYLASE

Several amino acid residues important for the action of porcine pancreatic α-amylase on starch were modified using specific reagents: histidine groups by photooxidation with rose bengal or by diethylpyrocarbonate; cysteine by dithiobis nitrobenzoic acid; tryptophan by N-bromosuccimide and tyrosine by hydroxyl acetylation with N-acetylimidazole. These modifications, with the exception of cysteine, reduced the amylase activity but none of them alone was able to alter significantly the inhibition of the enzyme by a purified black bean (Phaseolus vulgaris) amylase inhibitor. Only by photooxidation that can modify several groups at the same time was the inhibitor action eliminated. The results suggested that the histidine of the amylase active site and tyrosine of the substrate binding site are not important for binding to the bean inhibitor. The terminal sugars of the amylase inhibitor were identified as mannose and xylose. Periodate oxidation of the carbohydrate moiety caused total loss of activity. The treatment of the inhibitor with α-mannosidase did not alter its inhibitor action on α-amylase.     

THE MOLECULAR WEIGHT OF α-AMYLASE INHIBITOR FROM WHITE BEAN cv 858B (PHASEOLUS VULGARIS L.) IS 56 kDa, NOT 20 kDa

The molecular weights (M_rs) of α-amylase inhibitors (αAIs)fiom 18 (Ah) bean cultivars estimated by Superose 12 gelfiltration chromatography were 22–62% smaller than those determined by Sephadex G-100 gel filtration and by polyacrylamide gel electrophoresis (PAGE) methods. αAI-4 from WKB cultivar 858B was purified and the Mr was shown to be 51.0 kDa based on Sephadex G-100 gel filtration chromatography and by PAGE. A M_r for aAI-4 of 56.714 kDa was determined by laser-assisted time-of-flight mass spectrometry and appears to be the true M_r of the mature glycosylated active aAI-4. The results show that Superose 12 gelfiltration chromatography is not usefulfor M_r determination of some proteins. Sodium dodecyl sulfate electrophoresis (SDS-PAGE) showed that the 56.7 kDa aAI-4 molecule dissociated into 45.0, 33.6,15.2 and 12.4 kDa submolecules, with only the two small subunits, a and β, present at high SDS concentration. This provides evidence that the aAI-4 molecules composition is α₂β².     

ANTIOXIDANT ACTIVITY OF PHENOLIC FRACTIONS OF WHITE BEAN (PHASEOLUS VULGARIS)

An extract from white bean seeds was prepared using 80% (v/v) acetone. Four fractions (I-IV) -were separated from the crude extract on a Sephadex LH-20 column using methanol as the mobile phase. The antioxidant activity of fractions was investigated in a β-carotene-linoleate model system. For individual fractions, IV spectra were recorded and the content of total phenolics was determined. Fractions were also characterized based on the number of phenolic compounds, and their antioxidant activity determined by TLC analysis. The presence of caffeic, p-coumaric, ferulic, and sinapic acids in the form of free and estrified compounds was found in fraction IV. One dominant phenolic compound was present in fraction III after acid hydrolysis with a maximum absorption at 278 nm. Results of the β-carotene-linoleate model system indicated that antioxidant activity of separated fractions did not correlate exactly with their content of total phenolic compounds and were in the order of IV>III>II>I. Individual fractions contained several phnolic compounds as noted by TLC. Spots on the plates sprayed with a solution of p carotene-linoleate indicated that these compounds can act as natural antioxidants. Absorption maxima in the W spectra showed that jlavonoids, and not phenolic acids, were the main phenolic compounds present in the separated fractions.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF TWO PROTEINACEOUS α-AMYLASE INHIBITORS FROM TRITICALE

A total of six α-amylase inhibitory proteins (isoinhibitors) were extracted from triticale (Triticum X Secale) seeds and two of them were purified to homogeneity. The isoinhibitors were extracted by 70% ethanol and produced, by Sephadex G-100 chromatography, two peaks that exhibited α-amylase inhibitory activity. Further purification of the most active peak by DEAE-cellulose chromatography resulted in six active fractions. Two of them designated as TAI-5 and TAI-6, considered to be homogeneous by both acidic and alkaline electrophoresis, were partially characterized. The isoelectric points were 4.80 and 4.70, and the molecular weights 39, 200 and 29, 200, respectively. Under dissociating conditions the molecular weights were 13, 500 and 13, 000, suggesting that the isoinhibitors are composed of three and two subunits, respectively. Both isoinhibitors were stable at different pHs, relatively stable at 98C, and resistant to proteolysis by trypsin, chymotrypsin and pepsin. The optimum interaction pH for both isoinhibitors with human salivary amylase was 7.9. They exhibited specificity to human salivary and porcine pancreatic α-amylases, but had no inhibitory activity on Bacillus subtillis, Aspergillus oryzae and endogenous triticale α-amylases.     

PURIFICATION AND CHARACTERIZATION OF α-AMYLASE INHIBITORS IN WHEAT (TRITICUM AESTIVUM VAR. ANZA)

Three α-amylase inhibitors were purified to homogeneity from Anza wheat (Triticum aestivum var. Anza) by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE-cellulose, CM-ceillulose and Sephadex G-50. Homogeneity was determined by disc gel and isoelectric focusing electrophoresis and by sedimentation equilibrium centrifugation. The inhibitors are designated 0.19, 0.28 and 0.55 on basis of their relative electrophoretic mobilities on polyacrylamide gels. The 0.19, 0.28 and 0.55 inhibitors had molecular weights of 24,000, 18,500 and 30,000 by polyacrylamide gel electrophoresis with different gel concentrations while the former two were 29,000 and 14,500 by sedimentation equilibrium centrifugation, respectively. The molecular weight of the 0.55 inhibitor was not determined by centrifugation. The isoelectric points were 5.9, 5.2 and 4.2 for the 0.19, 0.28 and 0.55 inhibitors, respectively. The three inhibitors had similar amino acid compositions but differed significantly in amounts of lysine, arginine, histidine, alanine, valine and phenylalanine. The 0.19 inhibitor was active against human salivary and hog pancreatic α-amylases but inactive against Bacillus subtilis and Aspergillus oryzae α-amylases. The 0.28 inhibitor had very weak activity against only human salivary α-amylase. The 0.55 inhibitor had activity against only human salivary α-amylase. The 0.55 inhibitor appears to differ from all previously reported wheat α-amylase inhibitors while the 0.28 inhibitor (protein) is unique in having essentially no inhibitory activity.     

STABILITY OF TERTIARY STRUCTURE OF PHASEOLIN OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) AS LIMITING FACTOR IN PROTEOLYSIS

The major storage protein, phaseolin, of red kidney bean (Linden variety) was purified by ammonium sulfate fractionation between 3 and 4.1 M. It was composed of three subunits with MW 49,000, 45,000, and 42,000 and there were no disulfide bonds. Phaseolin was treated with heat (121°C, 15 min), pH (1.0, 7.5, 9.0), urea (2 to 10 M), guanidine (3 to 8.5 M) and alkali (0.02 N NaOH) at 35°C to destabilize the tertiary structure. Increase of in vitro proteolysis by the prior treatments was estimated with chymotrypsin, trypsin, pepsin and pronase. Native phaseolin was more resistant than Hammarsten casein to hydrolysis by chymotrypsin, trypsin, pepsin and pronase. An equimolar mixture of chymotrypsin and trypsin gave lower proteolysis than the sum of hydrolysis by each proteinase separately. Chymotrypsin hydrolysis products of phaseolin inhibited trypsin, and trypsin hydrolysis products of phaseolin inhibited chymotrypsin. All the treatments listed above enhanced in vitro proteolysis. Phaseolin treated with 0.02 M NaOH had higher hydrophobicity and higher absorbance between 240 and 350 nm than native phaseolin, indicating that its tertiary structure was destroyed. Autoclaving at pH 7.5, treatment with 8.5 M guanidine or with 0.02 M NaOH increased the extent of proteolysis of phaseolin to a similar or higher level than Hammarsten casein. Guanidine (8.5 M) destroyed the tertiary structure of phaseolin sufficiently that almost all susceptible bonds were hydrolyzed by pepsin, chymotrypsin and trypsin (and probably pronase).     

THE LECTINS AND LECTIN-LIKE PROTEINS OF TEPARY BEANS (PHASEOLUS ACUTIFOLIUS) AND TEPARYCOMMON BEAN (PHASEOLUS VULGARIS) HYBRIDS

High levels of lectin activity were found in sixty cultivated and ten wild tepary (Phaseolus acutifolius) accessions. No lectin deficient varieties were observed and all examples studied contained both the phytohemagglutinin-E and L-like lectins previously described (Pusztai et al. 1987). There appeared to be no obvious differences between the wild and cultivated forms of the tepary lectins and all teparies studied contained lectin-like proteins in addition to the tepary lectins. One of the lectin related proteins (40 Kdalton subunit) was present in all teparies and may be comparable to arcelin, a lectin found in certain wild accessions of Phaselus vulgaris. All wild teparies contained a lectin related polypeptide of about 34 Kdaltons which appears to distinguish the wild teparies from the cultivated forms. Three tepary-common bean hybrids were examined and one hybrid was found to be expressing both tepary and common bean lectin genes.     

PARTIAL CHARACTERIZATION OF TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM BEAN (PHASEOLUS VULGARIS L., VAR. ROSINHA G2): CHEMICAL AND PHYSICAL PROPERTIES

The three trypsin inhibitors A, B and C previously isolated from Brazilian pink bean (Phaseolus vulgaris L. var. Rosinha G2) had molecular weights of 18,200 to 18,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 20,000 by gel filtration on Sephadex G-100 and 20,400 by sucrose density gradient ultracentrifugation with a Stokes molecular radius of 20 Å, a frictional coefficient of 1.14, a diffusion coefficient of 10.7 × 10^?7 cm²s^?1, a partial specific volume of 0.69 cm³g^?1 and a molar absorptivity of 5.5 × 10³ M^?1 cm^?1 at 280 nm. All three inhibitors bound two moles of trypsin and one mole of chymotrypsin. The K_i values for trypsin were: A, 8.5 × 10^?10 M; B, 1.8 × 10^?10 M and C, 6.8 × 10^?10 M while for chymotrypsin they were: A, 4.4 × 10^?7 M; B, 2.8 × 10^?8 M and C, 3.0 × 10^?8 M. Reductive methylation caused loss of inhibitor activity of all three inhibitors against trypsin without significantly affecting inhibitor activity against chymotrypsin (with exception of inhibitor B), indicating that the inhibitors have lysine in binding site for trypsin. Partial reduction of the disulfide bonds caused loss of inhibitor activity against both trypsin and chymotrypsin with some regain of inhibitor activity following dialysis. Cyanogen bromide cleaved all three inhibitors into two fragments with significant retention of inhibitor activity. Cyanogen bromide-treated inhibitor B had nearly twice the original inhibitor activity against trypsin with no loss of inhibitor activity against chymotrypsin.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM MAISE (ZEA MAIZE)

α-Amylase inhibitor is presented in maize seeds. It is a protein as indicated by precipitation with ammonium sulfate and trichloroacetic acid, denaturation by heat, digestion with proteases and by dye-staining. It was purified to homogeneity by ammonium sulfate precipitation and Sephadex G-75 gel filtration. It had an apparent molecular weight of 29,600 and did not contain any carbohydrate. Its properties differed from those of previously reported α-amylase inhibitors, since it was active against α-amylase of maize, produced during germination as well as against Bacillus subtilis α-amylase. It was also active against α-amylase from the insects Tribolium castaneum, Sitophilus zeamais and Rhyzopertha dominica, but it was inactive against α-amylase from human saliva, hog pancreas, Aspergillus oryzae, wheat, rye, barley, triticale, and sorghum. It was stable for 5 min at 96°C at pH 7. Maximal inhibition required at least 10 min of preincubation with the enzyme at pH 6.8 and 257deg;C. Polyacrylamide gel electrophoresis gave three protein bands, but only one was obtained in S.D.S. and mercaptoethanol.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF TWO α-AMYLASE INHIBITORS FROM BLACK BEAN (Phaseolus vulgaris)1

Two α-amylase inhibitors from black bean (Phaseolus vulgaris) were purified to homogencity using ammonium sulfate fractionation, DEAE-Sephadex chromatography, phenyl-Sepharose hydrophobic interaction chromatography and gel filtration with Sephadex G-100. The inhibitors were designated I–1 and I–2 based on their order of elution from the phenyl-Sepharose column. Both inhibitors are mannose containing glycoproteins, composed of subunits; active against porcine pancreatic, human salivary, and insect α-amylases and inactive against bacterial, mold, and plant α-amylases. The inhibitors I-1 and I–2 have molecular weights of 49,000 and 47,000 and isoelectric points 4.93 and 4.86, respectively. Both inhibitors have similar amino acid compositions and are rich in aspartic acid, serine, glutamic acid, valine, and threonine and are low in sulfur containing amino acids. I–2 is more resistant to heat denaturation than I-1.     

PREPARATION AND CHARACTERIZATION OF α-AMYLASE IMMOBILIZED ON COLLAGEN MEMBRANES

Crystalline pancreatic α-amylase was codispersed with hide collagen at pH 4.0 and tanned to form a membrane which degraded starch. The optimum pH for the codispersed membrane preparation was at pH 7.0 in contrast to the soluble enzyme which was as active at pH 8.0 as at pH 7.0. The immobilized enzyme responded maximally to 0.22M chloride whereas 0.02M chloride gave optimum rates for the soluble enzyme. The immobilized enzyme resisted thermal inactivation better than the soluble α-amylase. Raising the temperatures from 30 ° to 50 °C produced a 500% increase in rate for the bound enzyme. It was also demonstrated that membranes retained greater activity when stored in starch solution than in water. The effect of glutaraldehyde concentration on membrane activity was also studied.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM RYE (SECALE CEREALE) FLOUR

An α-amylase inhibitor from rye (Secale cereale) flour has been purified to homogeneity by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE- and CM-cellulose. The isoelectric point was pH 5.8, and the molecular weight 28,000 by polyacrylamide gel electrophoresis with different gel concentrations and 27,000 by sedimentation equilibrium centrifugation. Under denaturating conditions the molecular weight was about 14,000, indicating two subunits identical in size. The inhibitor was active towards human salivary and hog pancreatic α-amylases but inactive towards Bacillus subtilis and Aspergillus oryzae α-amylases. The pH optimum for the reaction between the rye inhibitor and human salivary α-amylase was 6.0. The inhibitor did not change activity when exposed to pH 2 (0.01M HCl), but prolonged digestion by trypsin destroyed the inhibitor. The rye α-amylase inhibitor lost about 80% of its activity after 10 min at 100°C.     

NUTRITIONAL EVALUATION OF BEAN (PHASEOLUS VULGARIS) PROTEIN. IN VIVO VERSUS IN VITRO PROCEDURE

Digestibility (D), Biological Value (BV) and Net Protein Utilization (NPU) of whole bean flour and of the protein fractions glutelin (GLU), globulin (GLO), globulin GI (GI), albumin (ALB) and a protease inhibitor/lectin rich fraction (PIL) were determined, after autoclaving (121C, 15 min). For the whole bean flour both in vivo and in vitro procedures were used. For the in vivo assay a nitrogen balance with rats was performed. In vitro evaluation was based on the mean essential amino acid index (MEAAI) and in vitro protein digestibility. Results for the whole bean flour (BF) showed no statistical differences (p ≤ 0.01) between in vivo and in vitro techniques. In vitro digestibility ranged from 75–93% for the protein fractions and was 76% for the whole bean protein; biological value for the protein fractions ranged from 62–79%, and was 85% in the whole bean protein; calculated NPU ranged from 47–76% for the fractions, and was 65% for the whole bean protein.