Peter Z. Allen

Lactoperoxidase: V. Identification and isolation of lactoperoxidase from salivary gland

Abstract Bovine tissues were analyzed for the presence of lactoperoxidase by immunodiffusion analysis. A protein antigenically identical to lactoperoxidase was shown to be present in the sublingual, submaxillary, and parotid glands. An antigenically related but not identical protein was present in pig salivary glands. As the enzyme could not be detected in any of the other tissues employed, it could be concluded that if present, it represented less than 0.04% of the extracted protein. Most notable among the tissues which did not contain the enzyme, are the thyroid, uterus, and lymph node. A method is presented for the isolation of lactoperoxidase from the submaxillary gland. The enzyme is a major protein of this tissue, comprising between 1 and 2% of the wet weight of the tissue. The relationship of lactoperoxidase to peroxidase activity reported in other studies of the salivary gland is discussed. It is clear that the “salivary peroxidase” and “iodide peroxidase” can be attributed to lactoperoxidase.

Lactoperoxidase: VI. Immunochemical studies on lactoperoxidase from the milk of several species☆

Abstract The production of specific anti-enzyme in response to immunization with purified lactoperoxidase could be demonstrated by assays of peroxidase activity on the supernatant fluids and precipitates obtained from quantitative precipitin determinations. Spectrophotometric analysis of specific precipitates formed in antibody excess for a distinctive antigen marker showed all the hemoprotein added as antigen to be quantitatively precipitated by antibody. Lactoperoxidase isolated from the milk of ruminants such as the goat and sheep were examined for their cross reactivity with anti-bovine lactoperoxidase and found to be immunochemically indistinguishable from cows milk lactoperoxidase in their immunodiffusion and quantitative precipitin behavior. Bovine lactoperoxidase is immunologically distinct from bovine transferrin, ceruloplasmin, red protein, and lactollin as well as dog myeloperoxidase. Exposure of lactoperoxidase to proteolytic enzymes or 8 M urea does not result in any change detectable by immunodiffusion. The diffusion coefficient of bovine milk lactoperoxidase was estimated by immunodiffusion to be 5.2 × 10 −7 cm sec − .

Lactoperoxidase. IV. Immunological analysis of bovine lactoperoxidase preparations obtained by a simplified fractionation procedure

Abstract Antiserum to crude lactoperoxidase was employed in agar diffusion and immunoelectrophoretic analyses of fractions obtained in the isolation and purification of lactoperoxidase from cows milk by ion-exchange chromatography and gel filtration. The presence of seven antigenic components could be detected in a crude enzyme preparation by immunological analysis and their fate monitored during the purification procedure. Purified lactoperoxidase, prepared by ion-exchange chromatography and gel filtration, was found by immunological analysis to consist of a single antigenic component.

IMMUNOCHEMICAL STUDIES ON A PANOSYL-AZOPROTEIN CONJUGATE*

Abstract The trisaccharide panose, 6- O - α -d-glucopyranosyl-4- O - α -d-glucopyranosyl-d-glucose, coupled to bovine serum albumin (BSA) via an azophenyl linkage was used as an artificial antigen to immunize rabbits. Antisera rendered specific for the introduced haptenic grouping by absorption with whole calf serum were studied by immunodiffusion, quantitative precipitation and hapten inhibition. Anti-hapten showed a specificity directed against the introduced p -phenylazo- s -panoside group. Hapten inhibition assays employing various di- and triglucosides, including two structural isomers of panose, established the relative importance of an α-configuration and a nonreducing terminal (1,6)-(1,4) ordered sequence of glucoside bonds for optimal interaction with antibody. A heterogeneity in the specificity of antibodies formed in response to s-panosyl-BSA was evidenced from cross reactions obtained between anti-panoside and various sugar azoprotein conjugates.

Immunochemical studies on some immune systems involving ß(1,4) linked glucose

Abstract Rabbit antiserum to a BSA-cellobiose conjugate was examined by immunodiffusion and quantitative hapten inhibition employing the synthetic polyvalent hapten phloroglucinol triazophenyl-s- d -cellobioside as antigen. The isomeric series of s-linked glucobioses and mixed oligosaccharides possessing either terminal nonreducing or internal (subterminal) cellobiose were assayed for their ability to inhibit precipitation. Anticonjugate was more effectively inhibited by oligosaccharides possessing a terminal non-reducing cellobiose unit than by compounds with subterminal cellobiose groups. antibody could readily distinguish the s-(1,4) from the s-(1,3) and s-(1,6) glucosidic linkages. The portion of horse anti-VIII, precipitable by BSA-Cello was examined by hapten inhibition and p- nitrophenyl -s- d -cellobioside , cellotriose and cellotetraose found to be equipotent inhibitors of precipitation suggesting a specificity directed against the s- d -cellobiosyl or cellotriose unit.

Isomeric, anti-rhamnose antibodies having specificity for rhamnose-containing, streptococcal heteroglycans

L-Rhamnose (6-deoxy-L-mannose) is a constituent carbohydrate unit of microbial, immunogenic heteroglycans and lipopolysaccharides, and often functions as the immunodeterminant group of such immunogens. Two types of anti-rhamnose antibody have now been isolated by affinity chromatography of immune sera obtained from rabbits immunized with vaccines of Streptococcus mutans, strain KI-R, and Streptococcus pneumoniae, type 32. The antibodies of one type were directed at a glycan of L-rhamnose, D-glucose, and D-galactose in the cell wall of S. mutans, and those of the other type, against a capsular glycan of L-rhamnose and D-glucose from S. pneumoniae. The two types of anti-rhamnose antibody were immunologically distinct, and showed no reciprocal cross-reactivity. Additional properties of the two types of antibody were determined; thus, both types of antibody were of the IgG class of immunoglobulins, both possessed molecular weights of 1.45 X 10(5), and both consisted of multiple or isomeric forms.

Immunochemical studies on a laminaribiosyl-azoprotein conjugate☆

Abstract Laminaribiose ( 3-o-β- d -glucopyranosyl- d -glucose ) was coupled to bovine serum albumin (BSA) by an azophenyl linkage to provide the synthetic antigen BSA-p-phenylazo-β-laminaribioside. The behavior of antisera prepared in rabbits immunized with the β-laminaribiosyl conjugate was examined by immunodiffusion, quantitative precipitation and hapten inhibition. Anticonjugate absorbed with carrier protein showed the greatest reactivity with the homologous β-laminaribiosyl-BSA antigen, but also showed some cross precipitation with β-cellobiosyl, β-sophorosyl and β-gentiobiosyl-BSA conjugates. Glucobioses linked through the β-(1 → 2), β-(1 → 3), β-(1 → 4) and β-(1 → 6) positions, as well as laminaridextrins and tri and tetrasaccharides of β-linked glucose possessing a laminaribiose moiety either at a nonreducing end location or at a subterminal location, were assayed for their ability to inhibit antilaminaribioside precipitation. Hapten inhibition data showed anticonjugate a possess a high degree of specificity directed against the terminal nonreducing β-laminaribiosyl end group.

Some structural features of the antigenic groupings involved in the cross reaction of oat glucan with Type VIII horse antipneumococcal serum

Abstract Structural features of antigenic groupings of oat glucan involved in the cross reaction with Type VIII horese antipneumococcal serum were examined by quantitative hapten inhibition. Oligosaccharides of the cellodextrin series up to cellotetraose were examined and their molar inhibitory potency found to increase with the number of β(1→4) linked glucose units present. Employing mixed oligosaccharides as inhibitors possessing either terminal or subterminal β(1→4) linked glucose units, the cross reactive portion of anti-VIII could be shown to interact as well as terminal non-reducing cellodextrinyl groups. The pentameric structural unit of oat glucan possessing a sequence of four glucosyl residues joined through three consecutive β(1→4) linkages probably provides the internal groupings largely responsible for cross reactivity with anti-VIII.

Chemical and immunochemical studies on lipopolysaccharides from pyocin 103-sensitive and -resistant Neisseria gonorrhoeae

The chemical and immunochemical properties of lipopolysaccharides (LPS) isolated from pyocin 103-sensitive and -resistant Neisseria gonorrheae were investigated. Marked differences were found in immunochemical behavior of LPS from pyocin-sensitive gonococcal strain JW31 and its isogenic pyocin-resistant variant JW31R. JW31 LPS readily precipitated wheat-germ agglutinin, soybean lectin, and rabbit anti-Streptococcus faecalis or horse anti-type 14 pneumococcal antibody. In contrast, JW31R LPS precipitated only soybean lectin. The combining-site specificity of anti-S. faecalis cross-precipitated by JW31 LPS, or type 14 pneumococcal capsular polysaccharide, was examined by hapten inhibition, and lactose found to be the most potent inhibitor. Horse anti-pneumococcal type 14 antibodies, cross-precipitated by JW31 LPS and streptococcal lactose polymer, exhibited heterogeneity with respect to combining site specificity. Gel filtration of LPS-derived core oligosaccharide showed both strain JW31 and JW31 R to possess R-type lipopolysaccharide with cores having a Mr approximately 1800. JW31R LPS contains more galactose but less hexosamine than JW31 LPS. Both JW31 and JW31R core oligosaccharides possess D-glucosamine and D-galactosamine, probably N-acetylated, as the only nonreducing end-groups, and (1 leads to 4)-linked D-glucose residues. Chemical data support immunochemical findings which indicate that lactose units occur as a structural feature of JW31 gonococcal LPS.

Immunochemical studies on α-amylase: I. Effects of denaturing agents and proteolytic enzymes on the immunochemical reactivity of α-amylase from Aspergillus oryzae

Abstract Crystalline enzyme prepared from a crude enzyme concentrate of Aspergillus oryzae, and found to consist of a single antigenic component by various physicochemical and immunochemical methods, was used to produce rabbit antisera. The contribution of some aspects of the structure of Aspergillus α-amylase antigen to interaction with antienzyme was examined. Exposure of enzyme to concentrated urea solutions under various conditions resulted in preparations giving reactions of partial identity with native enzyme in immunodiffusion analysis. Quantitative precipitin determinations employing urea-treated enzyme show that only a partial loss of immunochemical reactivity occurs even with prolonged treatment. After treatment with EDTA and EDTA in the presence of 0.1 M mercaptoethanol the enzyme also showed changes in its immunochemical reactivity. A comparison of immunochemical behavior and specific activity of α-amylase before and after treatment with leucine aminopeptidase or carboxypeptidase suggests that neither N-terminal nor C-terminal amino acid residues play a significant role in enzymic or immunochemical reactivity.