Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Summary Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.In honour of Professor P. van Duijn     

Histochemical detection of sialic acid residues using periodate oxidation

Synopsis The use of low concentrations of periodate for the detection of sialic acid residues in tissue sections has been investigated. Oxidation of aqueous solutions of sugar glycosides with 0.4mm periodate revealed that sialic acid was oxidized more rapidly than other sugars found in glycoproteins. Sequential treatment of tissue sections with 0.4mm periodate for 30 min followed by Schiff's reagent stained sialic acid residues but other sugar components were not stained under these conditions.     

Detection of cellular sialic acid content using nitrobenzoxadiazole carbonyl-reactive chromophores

The selective ligation of hydrazine and amino-oxy compounds with carbonyls has gained popularity as a detection strategy with the recognition of aniline catalysis as a way to accelerate the labeling reaction in water. Aldehydes are a convenient functional group choice since there are few native aldehydes found at the cell surface. Aldehydes can be selectively introduced into sialic acid containing glycoproteins by treatment with dilute sodium periodate. Thus, the combination of periodate oxidation with aniline-catalyzed ligation (PAL) has become a viable method for detection of glycoconjugates on live cells. Herein we examine two fluorescent nitrobenzoxadiazole dyes for labeling of glycoproteins and cell surface glycoconjugates. We introduce a novel 4-aminooxy-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDAO) (5) fluorophore, and offer a comparison to commercial dyes including the known 4-hydrazino-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDH) (2) and Bodipy FL hydrazide. We confirm specificity for sialic acid moieties and that both dyes are suitable for in vitro and in vivo labeling studies using PAL and fluorescence spectroscopy. The dyes examined here are attractive labeling agents for microscopy, as they can be excited by a 488 nm laser line and can be made in a few synthetic steps. These carbonyl-reactive chromophores provide a one step alternative to avidin-biotin labeling strategies and simplify the detection of sialic acid in cells and glycoproteins.     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.     

The inactivation of the bovine J blood group substance by the periodate ion

1. Treatment of J-positive (Jcs) bovine erythrocytes with periodate (0.25 mmol/l final concentration, 1 hour, room temperature) has no effect on the J activity. Higher periodate concentrations cause spontaneous haemolyses. 2. Treatment of the lipids extracted from (and containing all J activity of) Jcs erythrocytes with periodate leads to a decrease of J activity even with lower periodate concentrations. 3. Treatment of the stroma prepared from Jcs erythrocytes with periodate demonstrated the relative stability of the J antigen up to 0.25 mmol/l periodate. At the same time the sialic acid concentration of stroma is reduced to about 13% of the initial concentration. 4. Desialylation of Jcs erythrocytes or Jcs stroma with sialidase does not affect the J activity thus confirming previous findings. On the other hand, the J activity of desialylated Jcs stroma is much more susceptible to periodate. 5. It is concluded that membrane-bound sialic acid shields the membrane-bound J antigen from being attacked by periodate.     

A Model System for Convenient Fluorescent Labeling of Sugar Chain in Taka-amylase A

A convenient detection of sugar chains in Taka-amylase A (TAA) was done by using 40 μg of enzyme, where a decrease in the UV absorption of NaIO₄ during the periodate oxidation reaction was monitored. The periodate-oxidized sugar chain was labeled with a fluorescent reagent, N-1-ethylenediaminonaphthalene (EDAN), by incubation at pH 9.5 and 30°C for 1 h. The excess EDAN was removed by either quenching with o-phthaladehyde or Bio-Gel P-2 gel adsorption. Among the peptide fragments prepared from the EDAN-labeled TAA, a fluorescent peptide corresponding to the sugar chain was distinguished by the ODS column. These results suggest that periodate oxidation and subsequent fluorescent labeling were useful for the sensitive analysis of various glycoprotein samples.     

An evaluation of tritium and fluorescence labelling combined with multi-detector SEC for the detection of carbonyl groups in polysaccharides

The carbonyl content of a pectic polysaccharide from Sphagnum papillosum (sphagnan) and periodate oxidised alginates was investigated using three different carbonyl labelling strategies combined with size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and on-line fluorescence or off-line tritium detection. The labelling strategies were tritium incorporation via NaB³H₄ reduction, and fluorescent labelling with carbazole carbonyl oxyamine (CCOA), or 2-aminobenzamide (2-AB), respectively. Carbonyl quantification was based on labelled pullulan, dextran and alginate standards possessing only the reducing end carbonyl group. As a result the carbonyl distribution in the polysaccharides could be determined. In sphagnan it was found that the carbonyl content increased with increasing molecular weight, whereas in periodate oxidised alginate the carbonyl content was as expected independent of the molecular weight. The methods proved useful for carbonyl detection in water soluble polysaccharides in general. The tritium incorporation method was preferred for alkali stable polysaccharides, while the CCOA method was most suitable for acid stable polysaccharides with low carbonyl content. The 2-AB method is applicable for all polysaccharides tested with varying carbonyl content; however, it lacks the ability to detect ketone functionalities.     

In vitro monitoring sub-nanogram amounts analgin in human urine by its inhibitory of the luminol-periodate chemiluminescence reaction using reagent immobilization release technique

A selective and sensitive as well as rapid chemiluminescence (CL) flow sensor for the determination of analgin is described. The analytical reagents involved in chemiluminescence reaction, luminol and periodate, were both immobilized on an anion-exchange column. The CL signals produced by the reaction between luminol and periodate, which were eluted from the column through water injection, were decreased in the presence of analgin. Analgin was sensed by measuring the decrement of CL intensity, and which was observed linear over the logarithm of analgin concentration range of 0.1 to 50.0 ng mL(-1), and the limit of detection was 0.04 ng mL(-1) (3ó). At a flow rate of 2.0 mL min(-1), including sampling and washing, the detection could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The proposed procedure was applied successfully in the monitoring of analgin in human urine samples without any pre-treatment process. It was found that the analgin concentration reached its maximum after being orally administrated for 4 h, and the analgin metabolism ratio in 10 h was 9.28% in the body of volunteers. The flow sensor offered reagentless procedures and remarkable stability in determination of analgin, and could be easily reused over 80 h.     

Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures.

Periodic acid (1% w/v) solvated by anhydrous dimethyl sulfoxide (DMSO) readily induced a strong Schiff reaction in a variety of structures containing polysaccharides, but not glycogen. With the increasing amounts of water added to DMSO, glycogen was also oxidized, while the selective localization of other polysaccharides remained unimpaired. Periodate, solvated in the anhydrous acetic acid-DMSO mixture, rapidly induced concomitant oxidation of nucin and glycogen-containing structures. Sodium bisulfite addition derivatives of carbonyls, induced by periodate oxidation in DMSO, were stained meta- and orthochromatically with toluidine blue at controlled pH. Certain metachromatic tissue components were strongly birefringent in polarized light in contrast to the identical structures oxidized by aqueous periodate. Marked differences in staining reactions elicited in identical structures by periodate in DMSO as compared with aqueous periodate suggest that DMSO-periodate method considerably enhances the range of histochemical oxidations by periodate.     

Remarks on the cis-aconitic anhydride method

Summary The reliability of cis-aconitic anhydride (CAZA) method in the histochemical detection of phospholipids was investigated. It was found, that the prescribed oxidative polymerization with cobalt chloride and sodium periodate of fixed sections is rather ineffective because the bulk of phosphlipids was detected in pooled solvents used in preparative steps. Even if residual phospholipids are revealed in sections the method is not recommended in practical histochemistry.     

The effect of sodium periodate treatment on the modulation of the sodium pump in low-potassium type (LK) sheep red cells by the L antigen

1. The action of sodium periodate and neuraminidase on active and passive K+ transport in low-potassium type (LK) sheep red cells was investigated in relation to the contribution of the Lp and Ll antigens. 2. Active K+ transport in LK sheep red cells was not affected by treatment with sodium periodate (2 mM), or with neuraminidase. 3. Passive K+ transport in LK sheep red cells was increased by sodium periodate treatment in a concentration-dependent manner. The increase was not Cl- dependent, and so differed from the increased passive K+ uptake resulting from N-ethylmaleimide treatment. 4. HK sheep red cells treated with sodium periodate showed small increases in passive K+ uptake, and N-ethylmaleimide treatment used sequentially with sodium periodate resulted in further small increases in passive K+ uptake. 5. In LK sheep red cells the stimulation of active K+ transport by anti-L was impaired by 50% in cells treated with sodium periodate (2 mM) and was slightly lowered in cells treated with neuraminidase. 6. In LK sheep red cells inhibition of passive K+ transport by anti-L was not impaired by sodium periodate treatment (2 mM), or by neuraminidase treatment.     

Periodate-induced transformation of human peripheral blood lymphocytes and the resultant oxidation of membrane sialyl, galactosyl and fucosyl residues

Oxidation of human peripheral mononuclear cells with sodium periodate results in lymphocyte activation. Period-date, at optimal mitogenic concentrations, oxidizes membrane sialyl residues (NeuNAc) essentially into the 7 carbon analogue (C7-NeuNAc). Fucosyl and galactosyl residues are also oxidized by periodate, since propane 1,2-diol and glycerol are isolated in acid hydrolysates of lymphocytes oxidized by periodate and reduced by tritiated borohydride. The neuraminidase pretreatment of lymphocytes induces a 40-50% decrease of their response to periodate. Neuraminidase treatment of 108 human peripheral lymphocytes liberated 9.6 microgram NeuNAc (31 nmol), representing 68.5% of the total content. The neuraminidase treatment dramatically enhances the recovery of glycerol in hydrolysates of lymphocytes treated successively with periodate and tritiated borohydride.     

Hydrolytic enzyme substrates. II. A new method for measuring periodate

This report describes an accurate and sensitive method for quantitatively measuring periodate concentration. The substances used to determine periodate are 4(p-nitrophenoxy)1,2-butanediol and 4(2,4-dinitrophenoxy)1,2-butanediol. These substances are readily oxidized by periodate yielding β-nitrophenoxy aldehydes which undergoes a facile β-elimination in base to yield the colored nitrophenolate ion. The concentration of the nitrophenolate ion is thus equivalent to the concentration of periodate. This report documents the validity of this reaction as an analytical method. The method was shown to be capable of accurately measuring periodate in concentrations as low as 10^?8M. Its value in biochemical analyses was demonstrated by quantitatively measuring the amount of periodate used to oxidize small quantities of adenosine 5′-phosphate, d-arabitol and d-glucose. Its accuracy, sensitivity and ease of use was shown by its utility in estimating the molecular weight of yeast transfer RNA using about 6 A₂₆₀ units of this material.     

The inactivation of the bovine J blood group substance by the periodate ion

• 1 Treatment of J-positive (JR) bovine erythrocytes with periodate (0.25 mmol/1 final concentration, 1 hour, room temperature) has no effect on the J activity. Higher periodate concentrations cause spontaneous haemolyses.
• 2 Treatment of the lipids extracted from (and containing all J activity of) J^cs erythrocytes with periodate leads to a decrease of J activity even with lower periodate concentrations.
• 3 Treatment of the stroma prepared from J^cs erythrocytes with periodate demonstrated the relative stability of the J antigen up to 0.25 mmol/l periodate. At the same time the sialic acid concentration of stroma is reduced to about 13 % of the initial concentration.
• 4 Desialylation of J^cs erythrocytes or J^cs stroma with sialidase does not affect the J activity thus confirming previous findings. On the other hand, the J activity of desialylated J^cs stroma is much more susceptible to periodate.
• 5 It is concluded that membrane-bound sialic acid shields the membrane-bound J antigen from being attacked by periodate.

     

Periodate oxidation of chitosans with different chemical compositions

Periodate oxidation of chitosans with different chemical compositions were investigated by determining the consumption of periodate consumed, and the amount of ammonia and formaldehyde liberated during the reaction. Oxidised chitosans were further characterised by size-exclusion chromatography with online multi-angle light scattering (SEC-MALLS) to obtain the molecular weight distributions, and by elemental analysis to obtain the N/C ratio. Chitosans became only partially oxidised by periodate, reaching degrees of oxidation around 0.5, when oxidising with excess periodate. Overconsumption of periodate is attributed to the extensive depolymerisation, which occurs concomitantly with the oxidation, thereby exposing novel reducing and non-reducing ends which consume additional periodate. Both the rate and extent of overoxidation, and the rate of depolymerisation decreased with increasing F(A). A chitosan-specific degradation mechanism is probably involved in the depolymerisation in addition to the general free-radical-mediated degradation.     

Inhibition of trypsin and chymotrypsin by thiols. Biphasic kinetics of reactivation and inhibition induced by sodium periodate addition.

Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.     

Reversible oxidative activation and inactivation of protein kinase C by the mitogen/tumor promoter periodate.

The oxidant mitogen/tumor promoter, periodate, was used to selectively modify either the regulatory domain or the catalytic domain of protein kinase C (PKC) to induce oxidative activation or inactivation of PKC, respectively. Periodate, at micromolar concentrations, modified the regulatory domain of PKC as determined by the loss of ability to stimulate kinase activity by Ca2+/phospholipid, and also by the loss of phorbol ester binding. This modification resulted in an increase in Ca2+/phospholipid-independent kinase activity (oxidative activation). However, at higher concentrations (greater than 100 microM) periodate also modified the catalytic domain, resulting in complete inactivation of PKC. The oxidative modification induced by low periodate concentrations (less than 0.5 mM) was completely reversed by a brief treatment with 2 mM dithiothreitol. In this aspect, the modification induced by periodate was different from that of the previously reported irreversible modification of PKC induced by H2O2. However, the inactivation of PKC induced by periodate at concentrations greater than 1 mM was not reversed by dithiothreitol. Among the phospholipids and ligands of the regulatory domain tested, only phosphatidylserine protected the regulatory domain from oxidative modification. In the presence of phosphatidylserine, the catalytic site was selectively modified by periodate, resulting in formation of a form of PKC that exhibited phorbol ester binding but not kinase activity. Both reversible and irreversible oxidative activation and inactivation of PKC also were observed in intact cells treated with periodate. Taken together these results suggest that periodate, by virtue of having a tetrahedral structure, binds to the phosphate-binding regions present within the phosphatidylserine-binding site of the regulatory domain and the ATP-binding site of the catalytic domain, and modifies the vicinal thiols present within these sites. This results in the formation of intramolecular disulfide bridge(s) within the regulatory domain or catalytic domain leading to either reversible activation or inactivation of PKC, respectively. Thus, oxidant mitogen/tumor promoters such as periodate may be able to bypass normal transmembrane signalling systems to directly activate pathways involved in cellular regulation.     

Immunochemical detection of glycosphingolipids on thin-layer chromatograms.

A sensitive immunochemical method was developed for the detection of glycosphingolipids on thin-layer chromatograms. The procedure involves oxidation of diol groups of glycosphingolipids with sodium periodate, derivatization of the formed aldehyde groups with digoxigenin-hydrazide, and reaction of the bound digoxigenin with an alkaline phosphatase-labeled polyclonal anti-digoxigenin antibody. The latter is detected by an insoluble indigo-like dye as a result of dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate. The detectability of all glycosphingolipid species was improved over that of the orcinol and resorcinol staining methods. Two nanograms of the standard gangliosides GM1, GD1A, and GT1 was detected, whereas the detection limit for short-chain neutral glycosphingolipids was in the range of 20-50 ng. Long-chain glycosphingolipids were detectable with a particularly high sensitivity. Selective staining of the gangliosides could be achieved by the use of low periodate concentrations.     

Direct enzyme immunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish

Saxitoxin was coupled to horseradish peroxidase via a novel adaptation of the periodate reaction. Based on polyclonal antibodies against saxitoxin, this conjugate was used for the development of two formats of direct enzyme immunoassay (EIA)–a microtitration enzyme-linked immunosorbent assay (ELISA) and a test strip EIA. The detection of saxitoxin without instrumentation by visual evaluation of the test strip EIA is described. The detection limits for saxitoxin were 7 pg/ml (0·35 pg/assay) in the ELISA and 200 pg/ml in the test strip EIA using visual evaluation. Employing a simple procedure of sample preparation, both ELISA and test strip EIA were applied to the analysis of shellfish. The detection limits for saxitoxin in shellfish tissue of the ELISA and the test strip assay were 3 and 4 ng/g, respectively.     

A spectrophotometric method for the microdetermination of periodate

1. A method is described for measuring the concentration of periodate over the range 0.2-20mum by adding 1,2-di-(p-dimethylaminophenyl)ethane-1,2-diol to a sample solution. Periodate cleaves this compound to from two molecules of p-dimethylaminobenzaldehyde, the extinction of which is then read at 352mmu. 2. The method has been used to follow the course of periodate oxidations of serine methyl ester, ribonuclease A and ribonuclease S-protein. Addition of the reagent stops further periodate reaction by reducing the remaining periodate to iodate. 3. The presence of protein does not interfere with the assay.