scholarly journals Active fragments obtained by cyanogen bromide cleavage of ovomucoid

1976 ◽  
Vol 155 (2) ◽  
pp. 345-351 ◽  
Author(s):  
J G. Beeley
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Cyanogen Bromide ◽  
Peptide Chain ◽  
Terminal Portion ◽  
Trypsin Inhibitory Activity ◽  
Methionine Residues ◽  
Active Fragments

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.

scholarly journals A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum)

1976 ◽  
Vol 157 (3) ◽  
pp. 745-751 ◽  
Author(s):  
P Smirnoff ◽  
S Khalef ◽  
Y Birk ◽  
S W Applebaum
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Polyacrylamide Gel ◽  
Molar Ratio ◽  
Chymotrypsin Inhibitor ◽  
Trypsin Inhibitory Activity ◽  
Hydrolysis Of

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.

Lima Bean Protease Inhibitor: Reduction and Reoxidation of the Disulfide Bonds and Their Reactivity in the Trypsin–inhibitor Complex

1973 ◽  
Vol 51 (7) ◽  
pp. 1021-1028 ◽  
Author(s):  
Frits C. Stevens ◽  
Elaine Doskoch
Keyword(s):  
Protease Inhibitor ◽  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Disulfide Bonds ◽  
Lima Bean ◽  
Air Oxidation ◽  
Complete Reduction ◽  
Inhibitor Complex ◽  
Molar Excess ◽  
Trypsin Inhibitory Activity

Lima bean protease inhibitor is a protein which inhibits both trypsin and chymotrypsin at different and independent sites. Complete reduction of the disulfide bonds of the inhibitor results in loss of biological activity. By air oxidation of the reduced inhibitor, full chymotrypsin inhibitory activity and up to 50% of the trypsin inhibitory activity can be regained; the rates at which these activities are regained were different. Attempts to obtain selective cleavage of one or a few disulfide bonds by carefully controlling reaction conditions were unsuccessful. All the disulfide bonds of lima bean inhibitor appear equally accessible to the reagent and with a less than twofold molar excess of dithioerythritol over the molarity of disulfide bonds, complete reduction was obtained; both inhibitory activities were equally sensitive to reduction and were lost as a linear function of the average number of disulfide bonds reduced and carboxymethylated. In contrast to this, the disulfide bonds of lima bean inhibitor are stabilized when the inhibitor is in the form of a molar complex with trypsin. Under these conditions, only one out of a possible eight disulfide bonds could be reduced in the inhibitor with up to a 10-fold molar excess of dithioerythritol. The modified inhibitor obtained after dissociation of reduced and carboxymethylated trypsin–inhibitor complex appeared fully active against both trypsin and chymotrypsin.

scholarly journals Anti-trypsin and antihypertensive activity of protein extracts from Nigella sativa seeds

2021 ◽  
Vol 58 (04) ◽  
pp. 1237-1243
Author(s):  
Bushra Javaid
Keyword(s):  
Protease Inhibitors ◽  
Ammonium Sulphate ◽  
Trypsin Inhibitor ◽  
Crude Extract ◽  
Inhibitory Activity ◽  
Nigella Sativa ◽  
Sulphate Concentration ◽  
Ammonium Sulphate Precipitation ◽  
Dpp Iv ◽  
Trypsin Inhibitory Activity

Protease inhibitors (PIs) are a ubiquitous, diverse group of molecules present in multiple forms in all organisms. These inhibitors inactivate proteases from predators/pathogens in addition to regulating intracellular proteolysis. In addition to intracellular localization, storage organs of plants are also a potential site of protease inhibitors. Proteins with trypsin inhibitory activity were isolated from Nigella sativa seed extracts by ammonium sulphate precipitation. Extraction conditions were optimized by choosing an optimum solvent, temperature and incubation period. The highest inhibitory activity of protein extracts was achieved by using 50 mM Tris buffer as solvent and overnight incubation of the suspension at 4°C. The crude seed extract fractionated at 60% ammonium sulphate concentration exhibited highest trypsin inhibitory activity, i.e., 60.15 ± 2.95 %, which was comparable to soybean trypsin inhibitor used as positive control. Ammonium sulphate precipitation of crude extract yielded 39.83-fold purification. Partially purified trypsin inhibitor exhibited 2.39±0.23 TIU mg-1. Additionally, Nigella sativa protein extracts were also investigated for their health-promoting effects against two important proteases, α- Dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE). Highest inhibitory activity against ACE was shown by the crude extract of N. sativa. Among AS fractions, 30% ammonium sulphate concentration exhibited highest inhibition activity against ACE and DPP-IV. Our results suggest that the widely believed role of N. sativa in control of hypertension may at least be partially shared by inhibition of ACE. This is the first study conducted to evaluate the biological activity of N. sativa protein extracts suggesting a potential use of N. sativa proteins in management of hypertension as well as an important source of trypsin inhibitor. Further identification, purification and characterization of different bioactive compounds from N. sativa are being carried out.

scholarly journals A Novel Kunitzin-Like Trypsin Inhibitor Isolated from Defensive Skin Secretion of Odorrana versabilis

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 254 ◽  
Author(s):  
Yanjing Dong ◽  
Daning Shi ◽  
Yuan Ying ◽  
Xinping Xi ◽  
Xiaoling Chen ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
The Novel ◽  
Skin Secretion ◽  
Amphibian Skin ◽  
Sequence Alignments ◽  
Molecular Weights ◽  
Trypsin Inhibitory Activity ◽  
Skin Secretions ◽  
Potent Inhibitory Activity

Protease inhibitors that were identified from amphibian skin secretions with low molecular weights and potent inhibitory activity were thought to be potential candidates for novel peptide drugs. Here, a novel peptide with trypsin inhibitory activity was found in the skin secretion of the Chinese bamboo leaf odorous frog, Odorrana versabilis. Based on the sequence alignments of sequencing results, the novel peptide (ALKYPFRCKAAFC) was named as Kunitzin-OV. The synthetic replicate of Kunitzin-OV was subjected to a series of functional assays, and it exhibited a trypsin inhibitory activity with a Ki value of 3.042 µM, whereas, when Lys-9 at P1 position was substituted by Phe, trypsin inhibitory activity was undetected and the chymotrypsin inhibitory activity was optimized with a Ki value of 2.874 µM. However, its protease-binding loop was catabolized by trypsin during the trypsin cleavage test. In conclusion, Kunizin-OV is a novel peptide with trypsin inhibitory activity as a member of kunitzins, which is a non-typical Kunitz-like trypsin inhibitor with a highly conserved reactive site (K-A) and quite a short sequence.

Permeability Enhancing and Chemotactic Activities of Lower Molecular Weight Degradation Products of Human Fibrinogen

1981 ◽  
Vol 45 (01) ◽  
pp. 090-094 ◽  
Author(s):  
Katsuo Sueishi ◽  
Shigeru Nanno ◽  
Kenzo Tanaka
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Electron Microscopic Observation ◽  
Chemotactic Activity ◽  
Lower Molecular Weight ◽  
Electron Microscopic ◽  
Human Fibrinogen ◽  
Rabbit Skin ◽  
Molecular Weights ◽  
Active Fragments

SummaryFibrinogen degradation products were investigated for leukocyte chemotactic activity and for enhancement of vascular permeability. Both activities increased progressively with plasmin digestion of fibrinogen. Active fragments were partially purified from 24 hr-plasmin digests. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D or E. Electron microscopic observation of the small blood vessels in rabbit skin correlated increased permeability with the formation of characteristic gaps between adjoining endothelial cells and their contraction.These findings suggest that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions characterized by deposits of fibrin and/or fibrinogen.

Amino acid sequence of cyanogen bromide fragment CB3 of hog pepsin

1981 ◽  
Vol 46 (3) ◽  
pp. 655-666
Author(s):  
Ladislav Morávek ◽  
Vladimír Kostka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cyanogen Bromide ◽  
Methionine Residues ◽  
Cyanogen Bromide Fragment

On the basis of the knowlidge of thermolytic, chymotryptic and substilisin peptides the amino acid sequence was determined of cyanogen bromide fragment CB3 representing the region between methionine residues I and II of pepsin: Thr-Gly-Ile-Leu-Gly-Tyr-Asp-Thr-Val-Gln-Val-Gly-Gly-Ile-Ser-Asp-Thr-Asn-Gln-Ile-Phe-Gly-Leu-Ser-Glu-Thr-Glu-Pro-Gly-Ser-Phe-Leu-Tyr-Tyr-Ala-Pro-Phe-Asp-Gly-Ile-Leu-Gly-Leu-Ala-Tyr-Pro-Ser-Ile-Ser-Ala-Ser-Gly-Ala-Thr-Pro-Val-Phe-Asp-Asn-Leu-Trp-Asp-Gln-Gly-Leu-Val-Ser-Gln-Asp-Leu-Phe-Ser-Val-Tyr-Leu-Ser-Ser-Asn-Asp-Asp-Ser-Gly-Ser-Val-Val-Leu-Leu-Gly-Gly-Ile-Asp-Ser-Ser-Tyr-Tyr-Thr-Gly-Ser-Leu-Asn-Trp-Val-Pro-Val-Ser-Val-Glu-Gly-Tyr-Trp-Gln-Ile-Thr-Leu-Asp-Ser-Ile-Thr-Met.

scholarly journals Novel Role for Albumin in Innate Immunity: Serum Albumin Inhibits the Growth of Blastomyces dermatitidis Yeast Form In Vitro

2003 ◽  
Vol 71 (11) ◽  
pp. 6648-6652 ◽  
Author(s):  
Steven Giles ◽  
Charles Czuprynski
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Mammalian Species ◽  
Blastomyces Dermatitidis ◽  
Low Molecular Weight ◽  
Rat Serum ◽  
Yeast Form ◽  
Domain Iii

ABSTRACT In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

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