Immunoenzyme method of sequential saturation for determining antigens using the model of insulin

The method for the determination of insulin by means of the enzyme immunoassay, based on the use of insulin-peroxidase conjugates, has been developed. In this assay the scheme of the successive saturation of the active sites of antibodies is used. The antigenic properties of two conjugates differing in the method of their preparation are compared. The conjugates were obtained by the covalent binding of peroxidase, oxidized in its carbohydrate component, with insulin (conjugate 1) or hexamethylene-diamine-modified insulin (conjugate 2). The conjugates represented a mixture of oligomers differing in their molecular weight. Conjugate 1 possessed higher affinity to antibodies and higher enzymatic activity than conjugate 2. The method for evaluating the quality of antisera to insulin used in the assay has been proposed. The time of the insulin assay is 5-16 hours, the limit of insulin detection is 5 microU/ml, the variation factor is 3-12%.     

Synthesis of immunoglobulin conjugates with dehydrogenases

Three methods of synthesis of immunoglobulin conjugates with malate, lactate and glucose-6-phosphate dehydrogenases, involving the sodium metaperiodate oxidation of immunoglobulin carbohydrate component, use of water-soluble carbodiimide and the one-step glutaraldehyde technique, were compared. The glutaraldehyde method was shown to give immunoglobulin-dehydrogenase conjugates with high catalytic and immunochemical activity, which may be useful for enzyme-immunoassay.     

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.     

Oxidation of indole-3-acetic Acid-amino Acid conjugates by horseradish peroxidase

The stability of 21 amino acid conjugates of indole-3-acetic acid (IAA) toward horseradish peroxidase (HRP) was studied. The IAA conjugates of Arg, Ile, Leu, Tyr, and Val were oxidized readily by peroxidase. Those of Ala, β-Ala, Asp, Cys, Gln, Glu, Gly, and Lys were not degraded and their recovery was above 92% after 1 hour incubation with HRP. A correlation between the stability of IAA conjugates toward peroxidase-catalyzed oxidation and the hydrophobicity of the amino acid moiety conjugated to IAA was demonstrated. Polar amino acid conjugates of IAA are more resistant to HRP-catalyzed oxidation.     

Chitooligosaccharide-Induced Activation of o-Phenylenediamine Oxidation by Wheat Seedlings in the Presence of Oxalic Acid

A method for determination of oxidation of phenolic compounds by intact wheat seedlings using o-phenylenediamine (OPD) was developed. The reaction is initiated by the addition of oxalic acid to the incubation medium. It is suggested that an endogenous peroxidase and hydrogen peroxide formed during oxidation of oxalic acid by endogenous oxalate oxidase are involved in OPD oxidation. Treatment of plants with chitooligosaccharides (1-10 mg/liter) with acetylation degree of 65% and molecular masses of 5-10 kD significantly activated OPD oxidation by wheat seedlings.     

Assessment of the composition and structure of covalent complexes of superoxide dismutase with aldehyde dextran by analytical ultracentrifugation]

Superoxide dismutase was covalently coupled wih aldehyde dextran, a polymeric carrier of molecular mass of 70 kDa. Modification produced an increase in the enzyme thermostability. Modified preparations retained a high specific activity. The composition of the thus obtained conjugates was analyzed by the ultracentrifugation and diffusion methods. The protein induced the destruction of aldehyde dextran, the enzyme being modified by its fragments. The presence of aldehyde dextran excess in the incubation medium promoted superoxide dismutase dissociation into individual subunits. At the enzyme/carrier ratio of 1:02 modification occurred as covalent coupling of the biocatalyst subunits and its one-point modification.     

Isolation and characterization of <Emphasis Type="Italic">Lentinus edodes</Emphasis> (Berk.) singer extracellular lectins

Lectin preparations have been isolated and purified from the culture liquid of the xylotrophic basidiomycete Lentinus edodes (Berk.) Singer [Lentinula edodes (Berk.) Pegler]. The culture of L. edodes F-249 synthesizes two extracellular lectins different in composition and physicochemical properties. Extracellular lectin L1 from L. edodes is a glycoprotein of mono-subunit structure with molecular weight of 43 kD. L1 is comprised of 10.5 +/- 1.0% (w/w) carbohydrates represented by glucose (Glc). Extracellular lectin L2 is a proteoglycan of mono-subunit structure with molecular weight of 37 kD. L2 is comprised of 90.3 +/- 1.0% (w/w) carbohydrates represented by Glc (73% of the total mass of the carbohydrate moiety of the lectin molecule) and galactose (Gal) (27% of the total mass of the carbohydrate part of the lectin molecule). The content of Asn in L2 is high, i.e. 42% (w/w) of total amino acids. This fact along with the composition of the carbohydrate part of the molecule (Glc + Gal) allows one to assign L2 to N-asparagine-bound proteins. Both lectins are specific to D-Gal and lactose (Lac) at an equal for L1 and L2 minimal inhibiting concentration of these carbohydrates (2.08 mM Gal and 8.33 mM Lac). Other carbohydrates to which the lectins show affinity are different for the two lectins: Rha (4.16 mM) for L1 and Ara (4.16 mM) and mannitol (8.33 mM) for L2. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans.     

Purification and characterization of human salivary peroxidase

Human salivary peroxidase (SPO) has been purified to homogeneity by subjecting human parotid saliva to immunoaffinity, cation exchange, and affinity chromatography. These procedures resulted in a 992-fold purification of the enzyme. When purified SPO was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), three Coomassie stainable bands were apparent, all of which stained positive for enzyme activity. The apparent molecular weights of the three bands were 78,000, 80,000, and 280,000 as analyzed by SDS-PAGE. Reduction with 2-mercaptoethanol resulted in a decreased mobility of these bands, and enzyme activity could no longer be detected on the gels. The SPO preparation had the characteristic peroxidase heme spectrum in the range 405-420 nm. The ratio between the absorbance of the Soret band (412 nm) and the absorbance at 280 nm was 0.81. The enzyme activity was inhibited by the classical peroxidase inhibitors cyanide and azide. Salivary peroxidase is similar to bovine lactoperoxidase (LPO) in amino acid composition, in ultraviolet and visible spectrum, in reaction with cyanide, in susceptibility to 2-mercaptoethanol inactivation, and in thermal stability. The two enzymes differ in carbohydrate composition and content. SPO contains 4.6% and LPO 7% total neutral sugars. The ratio of glucosamine to galactosamine is 2:1 in SPO and 3:1 in LPO. SPO contains mannose, fucose, and galactose in a molar ratio of 1.5:1.5:1.0, while the ratio was 14.9:0.5:1.0 in LPO. Glucose was present in both preparations in minor amounts. The concentration of azide required for 50% inhibition of enzyme activity was 20-fold greater for LPO than for SPO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides

The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.     

Chemoselective oxime and thiazolidine bond formation: a versatile and efficient route to the preparation of 3'-peptide-oligonucleotide conjugates

Oligonucleotides carrying an aldehyde moiety at the 3'-end were synthesized by the oxidation of a 1,2-diol precursor. These were coupled to peptides bearing a cysteine residue for thiazolidine formation and an aminooxy group for oxime formation. The conjugation reaction proved very efficient and selective, thereby allowing the preparation of 3'-peptide-oligonucleotide conjugates in good yield. The conjugation was achieved in aqueous solution without using any protection strategy. Moreover, the present approach neither requires the use of peptide in excess nor changes the hybridization properties of the conjugates.     

Separation of the bovine colostrum M-1 glycoprotein into two components

1. Two glycoproteins were isolated from the M-1 acid glycoprotein fraction of bovine colostrum. 2. The lighter glycoprotein had a molecular weight of 7200, contained about 28.4% of carbohydrate, and had an absorption maximum at 275nm. The heavier glycoprotein had a molecular weight of 12000, contained 39.0% of carbohydrate, and had no absorption maxima in the 240-300nm. range of the spectrum. 3. The carbohydrate moiety of both glycoproteins was removable from the polypeptide moiety under the conditions of the beta-elimination reaction. 4. Periodate oxidation experiments showed that sialic acid was linked to galactose in both proteins.     

Metabolism of Auxin in Tomato Fruit Tissue: Formation of High Molecular Weight Conjugates of Oxindole-3-Acetic Acid via the Oxidation of Indole-3-Acetylaspartic Acid

High performance liquid chromatography of extracts of tomato (Lycopersicon esculentum Mill.) incubated with a relatively low concentration (4 μm) of [1-¹⁴C]indole-3-acetic acid (IAA) revealed the presence of two major polar metabolites. Hydrolysis of the two metabolites with 7 n NaOH yielded the same compound, which had a retention time similar to that of ring-expanded oxindole-3-acetic acid (OxIAA) on high performance liquid chromatography. The identity of the indolic moiety of these conjugates as OxIAA was further confirmed by gas chromatography-mass spectrometry. Chromatography of the two OxIAA conjugates on a calibrated Bio-Gel P-2 column indicated that their molecular weights are about 1200 and 1000. Aspartic acid and glutamic acid were the major amino acids detected in acid hydrolysates of the two conjugates. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of indole-3-acetylaspartic acid (IAAsp) with a concomitant decrease in the formation of the two OxIAA conjugates. Feeding experiments with labeled IAAsp and OxIAA showed that IAAsp and not OxIAA is the precursor of these conjugates. The data obtained indicate that exogenous IAA is converted in tomato pericarp tissue to high molecular weight conjugates, presumably peptides, of OxIAA via the oxidation of IAAsp. The oxidation of IAAsp seems to be a rate-limiting step in the formation of these conjugates from exogenous IAA.     

Facile synthesis of stable and lectin-recognizable DNA-carbohydrate conjugates via diazo coupling

Stable and lectin-recognizable DNA-carbohydrate conjugates were prepared by diazo coupling of lactose and cellobiose derivatives to fragmented salmon testes DNA. The diazo coupling is suggested to take place selectively to guanine bases since the amount of lactose moiety introduced was directly proportional to the G content of various DNAs with different G contents. According to the CD spectra, the conjugates bearing carbohydrate less than 25% content kept a typical B-type conformation similar to native DNA. The conjugates possessed higher melting temperature and stronger nuclease resistance both to exo- and endonucleases than native DNA. Gel shift assay and fluorescence binding assay showed that the DNA-lactose conjugates were specifically bound to galactose-specific lectin RCA(120) with strong binding affinity (Ka = 10(4)-10(5) M(-1)) due to glycoside cluster effect. This facile method will be a useful protocol of molecular design for cell-targeted gene therapy.     

Comparative study of the extracellular laccases from Cerrena unicolor 059 0784 and Pleurotus oastreatus 0432

The laccases of the basidiomycetes Cerrena unicolor 059, C. unicolor 0784, and Pleurotus oastreatus 0432 were assayed comparatively. The laccases were isolated as homogenous preparations with molecular weight 55, 56, and 57 kD, respectively. The three enzymes were found to be glycoproteins. The carbohydrate moiety of the glycoproteins included mannose, galactose, and N-acetylglucosamine. The carbohydrate moiety of the laccases from C. unicolor 059, C. unicolor 0784, and P. oastreatus 0432 accounted for 17, 23, and 24%, respectively. The pH optimum of the enzymes was at 4.0, 3.75, and 5.6, respectively. Thermal stability testing of laccases at 40 degrees C revealed that the C. unicolor 0784 enzyme was characterized by the highest thermal stability (after 172-h incubation the enzyme activity was maintained at a level of 25%). The Michaelis constant (Km) values of the reactions of oxidation of pyrocatechol, hydroquinone, and potassium ferrocyanide catalyzed by the basidiomycete laccases were determined.     

Human serotonectin: a blood glycoprotein that binds serotonin. Chemical and physiological characterization

A glycoprotein that circulates in human blood, binds to the surface of platelets and white cells and also binds serotonin with high affinity and specificity has previously been purified and partially characterized. This glycoprotein has been called serotonectin. Antibodies raised against serotonectin inhibited the uptake of [3H]serotonin by platelets. We now report on the amino acid and carbohydrate composition of this protein as well as on some of the properties of the protein from which the carbohydrate moiety was removed. Serotonectin (apparent molecular weight 200 000; as judged by SDS-polyacrylamide gel electrophoresis) is an acidic protein that contains about 13% carbohydrate (w/w) consisting of mannose, galactose, glucosamine and sialic acid in a molar ratio of 2:1:4:0.8. Initial characterization suggests that serotonectin is a sialoglycoprotein of complex-type oligosaccharide N-linked to asparagine through N-acetylglucosamine. Treatment of serotonectin with neuraminidase resulted in a quantitative release of sialic acid without loss of antigenicity or binding capacity for [3H]serotonin. Treatment of desialylated serotonectin under non-denaturing conditions with almond glycopeptidase A resulted in 60-80% release of sugar. The protein moiety of the glycopeptidase-digested material showed no change in the capacity to bind [3H]serotonin and exhibited the same antigenic properties as untreated serotonectin. These data show the non-involvement of the carbohydrate moiety of human serotonectin in the mechanism of binding serotonin but the possible contribution of this moiety to a tighter interaction with the serotonectin receptor.     

Enzymes in Organic Synthesis VII—Enzymatic Activity of Hrp After Chemical Modification of the Carbohydrate Moiety

The carbohydrate moiety of horseradish peroxidase was conjugated with hexadecylamine or octylamine in a micellar medium. Recovery and purification of these conjugates was facilitated by the short length of the added spacers. The modification increased the liposolubility of the enzyme without detracting from its catalytic activity. For the hexadecylamine conjugate, the optimum reaction temperature was increased by 10d`C. In addition, activity in organic solvents, such as toluene or chloroform, remained high, even at 70d`C.     

Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.     

Structural studies on a glycopeptide from the tree fungus Cyttaria harioti Fischer

A glycopeptide (In₁) was isolated by phenol-water extraction from Cyttaria harioti Fischer, parasite of Nothofagus sps. Neutral sugars account for 89% of In₁ and were characterized as glucose, mannose, and galactose. Glucosamine, identified by g.l.c., was colorimetrically estimated (5.8%). The molar ratio of Glc:Man:Gal:GlcNAc was 17:11:3:2. The linkages between the various monosaccharide residues were established through methylation analysis and periodate oxidation studies. The anomeric configurations of the various glycosyl groups were determined by chromium trioxide oxidation of the acetylated polysaccharide. The results were confirmed by ¹³C-n.m.r. spectroscopy. The sugar chain is N-glycosyl-linked to the peptide. Structural features of the carbohydrate moiety of glycopeptide In₁ are described.     

A uniform approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A uniform approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with an aglycone moiety amino group can be coupled with PBC carboxyl derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a coupling agent. Both N- and O-glycosides differing in the conformational mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides. For example, not only oligosaccharides obtained by chemical synthesis but also reducing oligosaccharides isolated from natural sources can be transformed into N-glycosides. The approach was tested by the example of conjugation of the carboxyl derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand for lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Studies on pig serum lipoproteins. IV. Isolation and characterization of glycopeptides from pig serum low density lipoprotein.

Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.