Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds.

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.

The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).

Antinutritional factors in anasazi and other pinto beans (Phaseolus vulgaris L.).

Antinutritional factors of anasazi bean were compared to traditional pinto bean (Phaseolus vulgaris L.). Anasazi beans contained less (p<0.001) soluble and bound condensed tannins compared to pinto beans. No differences (p>0.05) in stachyose and raffinose content were found between the two bean types; verbascose was not detected at all. Significant (p<0.05) differences in lectin content were observed between anasazi and pinto bean. The lectins of anasazi beans were classified as non toxic and those of the pinto beans as toxic types. No differences (p>0.05) in inhibitor activity against human and bovine trypsin and chymotrypsin were found between the two bean types.

Primary structures of proteinase inhibitors from Phaseolus vulgaris var. nanus (cv. Borlotto).

The primary structures of two trypsin-chymotrypsin inhibitors from Phaseolus vulgaris var. nanus (bush bean, cv. Borlotto), PVI-3(2) und PVI-4, were derived from automated Edman degradation data, amino acid composition and manual Edman degradation results of enzymatic fragments and homology with other Bowman-Birk type proteinase inhibitors. The highest degrees of homology were observed between PVI-3(2) or PVI-4 and the trypsin-chymotrypsin inhibitors from lima beans (LBI I, IV and IV', 86%), black-eyed peas (BTCI, 81%), and, in part, adzuki beans (ABI I, II and II', 74-77%). Similarly, the primary structure of the trypsin-elastase inhibitor from the same source, PVI-3(1), was deduced which showed highest homology with that of the trypsin-elastase inhibitor GBI II from garden beans (92%), followed by GBI II' from garden beans (86%) and C-II from soybeans (71%). In contrast, homology between PVI-3(2) and PVI-4 on the one hand and PVI-3(1) on the other was relatively low (61%).

Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis.

Fluorogenic substrates were tested as a means of increasing both the sensitivity and the selectivity of trypsin and chymotrypsin inhibitor detection after electrophoretic separation. Out of six substrates applied to cellulose acetate membranes, N alpha-benzyloxycarbonyl-L-arginine-4-methylcoumarinyl-7-amide (Z-Arg-MCA) and benzyloxycarbonyl-glycyl-glycyl-L-arginine-4-trifluoromethylcoumariny l-7-amide (Z-Gly-Gly-Arg-TFMCA) were found to be suitable for trypsin, and L-alanyl-L-alanyl-L-phenylalanine-4-methylcoumarinyl-7-amide (Ala-Ala-Phe-MCA) was suitable for chymotrypsin. A procedure to detect trypsin and chymotrypsin inhibitors, and to discriminate between them, was developed. After electrophoresis, slab gels were first incubated with the enzyme (bovine trypsin, bovine chymotrypsin, or human duodenal juice) at 37 degrees C, and then covered with the respective substrate membrane and incubated at room temperature while being observed under UV light. Dark blue inhibitor bands on a light-blue-fluorescent background were obtained with Z-Arg-MCA/trypsin and Ala-Ala-Phe-MCA/chymotrypsin, whereas Z-Gly-Gly-Arg-TFMCA/trypsin resulted in dark inhibitor bands on a fluorescent green background. The "inhibitor overlay membrane technique" (IOM technique) was used after polyacrylamide gel isoelectric focusing with carrier ampholytes and immobilized pH gradients, pore-gradient polyacrylamide gel electrophoresis, and sodium dodecyl sulfate pore-gradient polyacrylamide gel electrophoresis.

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Reaction with the human and bovine proteinases.

The reaction between the three Bowman-Birk proteinase inhibitors isolated from fenugreek seeds (TFI-B2, TFI-N2 and TFI-A8) and the human and bovine proteinases was investigated by studying the complexes formed and their properties. TFI-B2, the Lys-Leu trypsin chymotrypsin inhibitor, can bind 1.9 mol human trypsin (HT), 1.3 mol bovine trypsin (BT) and/or 0.4 mol human (HCT) or bovine (BCT) chymotrypsin per mole of inhibitor. HT was bound at the two reactive sites and BT mainly at the lysine-containing trypsin-reactive site, whereas HCT and BCT were only bound at the leucine-containing chymotrypsin-reactive site. TFI-N2, the Arg-Leu trypsin chymotrypsin inhibitor, could bind 1 mol BT and BCT, but 1.3 mol HT and 1.2 mol HCT per mole of inhibitor. In addition to the usual binding, the human enzymes could also be bound at the respective "wrong" reactive site. TFI-A8, the Arg-Arg trypsin inhibitor, binds 2 mol HT or BT per mole of inhibitor at the two trypsin-reactive sites, whereas HCT and BCT (about 0.2 mol/mol) are bound to one of the two "wrong" reactive sites.

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Reactive sites and C-terminal sequences.

The reactive sites and the C-terminal sequences of three trypsin chymotrypsin inhibitors from fenugreek seeds (TFI-B2, TFI-N2, and TFI-A8) were determined by chemical modification and carboxypeptidase degradation of native und enzymatically modified inhibitors. TFI-B2 contained lysine and leucine in the trypsin- and chymotrypsin-reactive sites, respectively, and -(Lys)-Phe-Leu-Ile was the C-terminal sequence. TFI-N2 possessed arginine and leucine in the trypsin- and chymotrypsin-reactive sites, respectively, and -(Tyr)-Lys-Ile-Leu at the C-terminus. TFI-A8 contained two arginines, one in each of the two reactive sites. At least one of these sites, although mainly directed against trypsin, could also bind some chymotrypsin. -(Leu)-Phe-Ile-Arg was found to be the C-terminus in TFI-A8. These results confirmed that all three fenugreek inhibitors belong to the Bowman-Birk proteinase inhibitor family.

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Isolation and characterization.

Three fenugreek inhibitors (TFI-A8, TFI-N2, and TFI-B2) were isolated from an inhibitor preparation by anion exchange chromatography and subsequent preparative isoelectric focusing using immobilized pH gradients and the canal technique. The purified inhibitors inhibited the enzymes tested differently: TFI-A8 exhibited a high inhibition of trypsin (8.2 mg human trypsin/mg and 8.1 mg bovine trypsin/mg) and a very low inhibition of chymotrypsin (0.8 mg human chymotrypsin/mg and 1.0 mg bovine chymotrypsin/mg). TFI-N2 inhibited the four enzymes to about the same extent (5.0 mg/mg human and 4.1 mg/mg bovine trypsin; 4.9 mg/mg human and 3.7 mg/mg bovine chymotrypsin). TFI-B2 displayed a high inhibition of trypsin (7.5 mg/mg human and 5.1 mg/mg bovine) and a low inhibition of chymotrypsin (1.8 mg/mg human and 1.9 mg/mg bovine). On average, the human enzymes were inhibited better than the bovine ones by the purified inhibitors. The inhibitors contained high amounts of cystine (five or six disulfide bridges per molecule), aspartic acid, threonine, serine and proline, no valine and methionine and two of them also no tryptophan. Their molecular masses were about 6 kDa. Their inclusion into the Bowman-Birk soybean proteinase inhibitor family is discussed.

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Demonstration and purification.

Fenugreek contained proteinase inhibitors inhibiting 5-9 mg human trypsin, 5-7 mg bovine trypsin, 2-6 mg human chymotrypsin, and 1-3 mg bovine chymotrypsin per g seed material. About 30 inhibitors were electrophoretically detected, and 23 of them, inhibiting all the four enzymes, were characterized by means of their isoelectric points: a group of acid inhibitors (TFI-A1 to A10, pI 4.48-5.12), a group of neutral inhibitors (TFI-N1 to -N6, pI 5.91-6.71), and a group of basic inhibitors (TFI-B1 to -B7, pI 7.76-9.77). To eliminate the galactomannans which complicate further purification, coarsely ground seeds were separated by density into two fractions, seed coats + endosperm and cotyledons + embryos (C + E). Isolation of the fenugreek inhibitors by extraction of fraction C + E, followed by ammonium sulfate fractionation and affinity chromatography on anhydrotrypsin-Sepharose, resulted in an about 700-fold enrichment.

Model studies on the heating of food proteins. Amino acid composition of lysozyme, ribonuclease and insulin after dry heating.

Lysozyme, ribonuclease and insulin were exposed to dry heating for 1 to 24 h at temperatures between 80 and 180 degrees C. Amino acid analyses of the heated samples showed that most of the amino acids are stable up to 120 degrees C. Initially, at higher temperatures, an almost rectilinear decrease took place which reached a critical stage at 160 degrees C. Nonpolar aliphatic, acidic and aromatic amino acids were all relatively stable (maximum loss less than 20% after 24 h at 180 degrees C). The lability of the other amino acids increased in the order proline, arginine, histidine, cysteine, threonine, lysine, tryptophan, serine, and methionine. Methionine was 86% decomposed after 24 h at 180 degrees C. Loss of trinitrobenzene sulfonic acid-reactive lysine ("available lysine") reached 20% at 100 degrees C and essentially 100% after 24 h at 180 degrees C. Maximum loss in weight during heating was 11%, although maximum protein loss was between 20 and 35%. Reaction orders and activation energies were estimated for some of the amino acid losses. Of the atypical amino acids ("hot spots") lysinoalanine, allo-isoleucine and ornithine that were detected, only lysinoalanine is useful as an indicator to detect amino acid damage after dry heating.

[Chemical composition of seeds from Cucumeropsis mannii Naudin and their suitability as food]. / Beitrag zur chemischen Zusammensetzung der Samen von Cucumeropsis Mannii Naudin und zu ihrer Eignung als Lebensmittel.

Dehulled seeds from Cucumeropsis Mannii Naudin mainly consist of lipids (40.3%) and proteins (34.5%). Carbohydrates, minerals, and water amount to 16.5, 3.7, and 5.9%, resp. From this composition a caloric value of 2 190 kJ/100 g is calculated. The major component of the oil linoleic acid (57.9%). Short-chain fatty acids are absent. All important macro and micro nutrient elements are present in sufficient amounts for human nutrition. The seeds are rich in vitamin E and in niacin (30.8 mg/100 g and 14.3 mg/100 g. resp.). Consumption of 100 g dehulled seeds covers the daily requirement of essential fatty acids, vitamin E and essential amino acids--methionine excepted. Besides starch (14.3 g/100 g) sucrose (1.14 g/100 g), raffinose (0.42 g/100 g) and stachyose (0.41 g/100 g) as well as traces of glucose and fructose are present. The proteins extracted with various solvents (H2O, 0.1 M KCl-, 0.1 mM K3PO4-, and 0.5% SDS-solution) are studied by amino acid analysis. SDS-electrophoresis and isoelectric focusing. Molecular weights of these proteins are between 5,000 and 80,000 daltons with the fraction between 20,000 and 35,000 predominating. The seeds exhibit weak inhibition of trypsin and do not inhibit alpha-chymotrypsin.

Comparative studies of the inhibitory action of some legume seeds, potato tubers, and bran against human and bovine proteinases.

Inhibition of human trypsin and chymotrypsin by proteinase inhibitors of plant origin was studied using the juice from the small intestine, without separation of the two enzymes, and synthetic amide substrates (BAPA, GLUPHEPA). The results were compared with the inhibition of the corresponding bovine enzymes. Extracts and preparations from legume seeds (13 Papilionoideae, 2 Caesalpinioideae, 3 Mimosoideae), potato tubes and bran were used as inhibitors; 9 of the seed extracts studied inhibited human chymotrypsin more and human trypsin less than the bovine enzymes. In similar tests 5 seed extracts inhibited the two human enzymes more than the bovine enzymes, whilst only one extract inhibited the two human enzymes less than the two bovine enzymes, and another inhibited human trypsin more and human chymotrypsin less than the bovine enzymes. In particular, human chymotrypsin was between two and twelve times as strongly inhibited as the bovine enzyme by some two thirds of the species studied, sometimes exhibiting chymotrypsin-inhibitory activities usually observed only with trypsin. Inhibitor preparations from the above legumes and two non-leguminous plant foods exhibited similar results.

Isolation and characterization of some proteinase inhibitors from Phaseolus vulgaris var. nanus.

Six proteinase inhibitors have been isolated from a crude inhibitor preparation from Phaseolus vulgaris var. nanus (bush bean: Borlotto) by gel chromatography and ion exchange chromatography. The isoelectric points of the inhibitors are between 4.35 and 5.65. The molecular weights of the inhibitors PVI-2, -3(1), -3(2), and -4 and between 8000 and 9500. The C-terminal and N-terminal amino acid residues and the amino acid compositions of these four inhibitors are given. The inhibitors PVI-1, -2, -3(1), -3(2), -4, and 5(1) all inhibit trypsin and with the exception of PVI-3(1) also alpha-chymotrypsin. PVI-3(1) inhibit elastase. The inhibitor mixture, PVI-G, exhibits a weak inhibition of some microbial serine proteinases. Some other endopeptidases and exopeptidases tested are not inhibited. Crude inhibitor preparations from P. coccineus and Pisum sativum show the same behaviour.

Model studies on the heating of food proteins--heat-induced oligomerisation of ribonuclease.

Commercial ribonuclease was heated at temperatures between 80 degrees C and 180 degrees C for 1--24 h. A stoichiometric oligomerisation was observed. Dimeric ribonuclease appeared after 1 h at 80 degrees C. With increasing time and/or temperature the number of oligomers formed rose at first. The highest oligomer that could be detected was the hexamer (140 degrees C, 8 and 16 h). A further increase in time and/or temperature resulted in a decrease of the number of oligomers and a rise in the polymer fraction. The importance of this reaction in the changes produced by heating food proteins and various reaction mechanisms are discussed.

[Comparative studies on the reactive sites against trypsin of some inhibitors from phaseolus coccineus and phaseolus vulgaris (author's transl)]. / Vergleichende Untersuchungen der trypsinspezifischen reaktiven Zentren verschiedener Phaseolus coccineus- und Phaseioul vulgaris-Inhibitoren

Three of the trypsin chymotrypsin inhibitors from the seeds of runner beans (Phaseolus coccineus L.), PCI 3,4(2), and 5, and three of the inhibitors from the seeds of french beans (Phaseolus vulgaris var. nanus), PVI 3, 4, and 5, contain a lysine residue in the reactive site against trypsin. One of the inhibitors from Phaseolus coccineus, PCI 2, contains an arginine residue there. All seven Phaseolus inhibitors investigated are double headed.

[Trypsin and chymotrypsin inhibitors in leguminosae VII. Partial amino acid sequences of the trypsin chymotrypsin inhibitor PCI 3 from Phaseolus coccineus (author's transl)]. / Trypsin- und Chymotrypsin-inhibitoren in Leguminosen VII. Partialsequenzen des Trypsin-Chymotrypsin-Inhibitors PCI 3 aus Phaseolus coccineus

The sequences of amino acid residues at the amino and carboxyl terminus and around the reactive sites of the trypsin chymotrypsin inhibitor PCI 3 from the seeds of runner beans (Phaseolus coccineus L.) were estimated by aminopeptidase O and carbosypeptidase A degradation before and after enzymatical modification with trypsin or chymotrypsin. Beginning at the amino terminus the sequences are :Ser-Glu-Ala-Gly-Gln-...,...-Ile-Tyr-Lys-Ser-Gln-(Pro)-...with Lys-Ser as reactive site against trypsin, ...-Asp-Val-Ala-Leu-Ser-(Pro)-...with Leu-Ser as reactive site against alpha-chymotrypsin, and ...-Thr-Arg-Ala-Lys-Phe-Leu as C-terminus. The importance of the serine residue in the reactive sites concerning the specificity of inhibitors is discussed.