alpha-chymotrypsin and metaperiodate

A contributing factor to food allergen stability is heat resistance. Peanut allergens in particular are resistant to heat, which results in their decreased solubility upon routine extraction and may have a profound influence on their continued presence in the digestive tract. Although there have been a number of studies characterizing soluble extracts of raw and roasted proteins, the relative solubility of the insoluble material following routine extraction for residual allergen characterization has not been investigated. The effects of various treatments on the re-solubilization and subsequent allergenicity of this insoluble peanut protein material are presented here.. Various methods to resolubilize the insoluble protein material were used, including pH, proteases and glycosidases. Protease digestion of nonextractable peanut proteins with pepsin, chymotrypsin and trypsin was performed in appropriate buffers as previously optimized for peanut proteins. Glycosidase activity in the presence of protease inhibitors was performed at pH 2. Digested samples were then subjected to SDS-PAGE/Western blot analysis using serum IgE from peanut-sensitive individuals.. Progressive roasting of peanuts resulted in a significant decrease in protein solubility. The acidic proteins were resolubilized moderately at high pH, with solubility decreasing as pH approached the pI of the protein. However, at pH 2 the solubility increased dramatically. More extensive resolubilzation was observed with amylase treatment, presumably due to cleavage of glycoside of glycoproteins. The protein released into solution had a high IgE-binding capacity. While amylase was effective at resolubilizing this material, digestive tract proteases were not.. The presence of these insolubilized peanut proteins provides a continuous source of major allergens to the gastrointestinal mucosal immune system.

Topics: Allergens; Amylases; Arachis; Chymotrypsin; Hot Temperature; Hydrogen-Ion Concentration; Immunoglobulin E; Oxidation-Reduction; Peanut Hypersensitivity; Pepsin A; Periodic Acid; Solubility; Trypsin

Heparin cofactor II (HC II) is known as a bifunctional inhibitor inactivating trypsin- and chymotrypsin type proteases. Its inhibitory activity increases in the presence of heparin, dermatan sulfate and chondroitin E. In the present study the inhibitory activity of HC II was investigated as function of various dermatan sulfate fractions and its stability was tested against oxidation reagents similar to thus secreted by activated leucocytes. High affinity dermatan sulfate (DS) increased the antithrombin inhibition activity of HC II about 1000-fold in contrast to about 100-fold in the case of low affinity DS. Oxidation of HC II carbohydrate side chains with sodium periodate showed less inactivation effects than oxidation by chloramine T or ammonium peroxodisulfate.

Topics: Chloramines; Chymotrypsin; Dermatan Sulfate; Heparin Cofactor II; Periodic Acid; Thrombin; Tosyl Compounds

The effect of sodium periodate and proteolytic enzyme treatments on the antibody binding capacity of Mycobacterium tuberculosis antigen (Ag) was studied by ELISA. Treatment with sodium periodate resulted in a marked decrease in the capacity of M. tuberculosis Ag to bind antibodies in human TB sera, but had no effect on the reactivity with antibodies in mouse. In contrast, treatment with proteolytic enzymes (trypsin and chymotrypsin) had no effect on the reactivity of M. tuberculosis Ag with human TB sera but reduced substantially the reactivity to antibodies in mouse antisera. These results indicate that anti-M. tuberculosis antibodies in human TB sera react predominantly with carbohydrate determinants and not with protein epitopes sensitive to trypsin and chymotrypsin. The bulk of murine antibodies on the other hand were directed against protein determinants and not the carbohydrate epitopes.

Topics: Animals; Antibodies, Bacterial; Antigen-Antibody Reactions; Antigens, Bacterial; Bacterial Proteins; Carbohydrates; Chymotrypsin; Enzyme-Linked Immunosorbent Assay; Epitopes; Immune Sera; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mycobacterium tuberculosis; Periodic Acid; Sonication; Trypsin; Tuberculosis

Hybrid enzymes which have two different enzyme activities linked together covalently may be useful reagents for various applications, such as the determination of complex biological structures. The present paper describes the preparation and purification of two such enzyme-enzyme conjugates, namely, trypsin-chymotrypsin and trypsin-alkaline phosphatase. Whereas the former has been prepared by using the well-known bifunctional reagent glutaraldehyde, the latter exploited the Schiff base formation between the oxidized carbohydrate moiety of alkaline phosphatase and the free amino groups of trypsin.

Topics: Alkaline Phosphatase; Animals; Cattle; Chymotrypsin; Dimethyl Adipimidate; Glutaral; Immunoenzyme Techniques; Methods; Periodic Acid; Trypsin

The phenomenon of glioma killing by lymphokine activated killer cells (LAK) was studied. We demonstrate that LAK cells generated by culturing the lymphokine interleukin-2 (IL-2) with peripheral blood lymphocytes from brain tumour patients destroys autologous glioma. The rat 9L glioma model was used to show that LAK killing was tumour-selective as glioma but not syngeneic normal brain tissue was destroyed. The susceptibility of both human and 9L rat glioma to LAK cell killing was markedly diminished by pretreating glioma cells with trypsin or chymotrypsin, but was unaffected by pretreatment with neuraminidase, glycosidases, sodium periodate or hydrocortisone. These results suggest that the cell surface determinant on glioma cells responsible for its tumour selective lysis by LAK is a protein sensitive to trypsin and chymotrypsin. The tumour-selective killing of glioma by LAK in vitro prompted the initiation of a Phase I study in which ten patients with malignant glioma have been treated with direct intracerebral injection of IL-2 or LAK without evidence of systemic or brain toxicity.

Topics: Adult; Aged; Animals; Borohydrides; Brain Neoplasms; Cell Line; Chymotrypsin; Cytotoxicity, Immunologic; Disease Models, Animal; Female; Glioma; Humans; Interleukin-2; Killer Cells, Natural; Lymphocyte Activation; Male; Middle Aged; Periodic Acid; Rats; Rats, Inbred F344; Trypsin

1. The reaction of alpha-chymotrypsin with sodium periodate at pH5.0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K(m) values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.

Topics: Acylation; Amides; Amino Acids; Biochemical Phenomena; Biochemistry; Catalysis; Chymotrypsin; Imidazoles; Kinetics; Methionine; Nitrophenols; Periodic Acid; Research; Spectrophotometry; Tryptophan; Tyrosine; Ultracentrifugation; Valine