New international F.I.P. method for the determination of the activity of Aspergillus oryzae proteases.

Proteases of Aspergillus oryzae are used as a drug in the therapy of digestive disorders. To standardize these enzyme the Enzyme Commission of the Fédération Internationale Pharmaceutique (F.I.P.) has tested a new determination method, which will be described below. The standard preparation of a mixture of Aspergillus oryzae proteases used in this test is characterized.

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.