Influence of Protein Modification and Glycosylation in the Catalytic Hydrogen Evolution Reaction of Avidin and Neutravidin: An Electrochemical Analysis.

To investigate glycans' influence on the behavior of glycoproteins on charged surfaces, avidin and its nonglycosylated and neutralized version neutravidin were studied by label-free chronopotentiometric stripping (CPS) analysis and alternating current voltammetry combined with a mercury electrode. Despite neutravidin's and avidin's similar size and structure, their CPS responses differed due to the different amounts of catalytically active free amino groups of lysine and arginine residues. Acetylation of the proteins resulted in the suppression of their CPS responses by almost four times for avidin and by about 50 % for neutravidin, respectively. On the other hand, the presence of glycans in the acetylated avidin induced about 30 % higher chronopotentiometric response compared to the acetylated neutravidin. We suggest that the presence, size and composition of the glycans influenced the CPS signal due to differences in the orientation at a charged surface. The obtained results can be utilized in glycoprotein research.

Cyclic and square wave voltammetry of chitooligosaccharides modified by osmium(VI) tetramethylethylenediamine.

Compounds containing vicinal diol (glycol) groups, including saccharides, could be modified with sixvalent osmium complexes with nitrogenous ligands, particularly with N,N,N',N'-tetramethylethylenediamine (Os(VI)tem). The modification products are electrochemically active. Here we show that aminosaccharides can also be modified by Os(VI)tem. We studied chitosan oligosaccharides in their acetylated and deacetylated form in 0.2 M Na-phosphate, pH 6.9. Deacetylated chitosan oligosaccharides with free amino groups modified by Os(VI)tem yielded two peaks (peak I' at -0.15 V and peak II' at about -0.38 V) despite the fact that these oligomers contain only one glycol group on the non-reducing end of the molecule. The electrochemical behavior of Os(VI)tem modified deacetylated chitosan oligomers differs from Os(VI)tem modified simple saccharides, containing only glycol groups, predominantly in peak I'. Our results suggest that free amino groups are involved in Os(VI)tem modification of chitosan oligomers.

Vicinal Diol-Tethered Nucleobases as Targets for DNA Redox Labeling with Osmate Complexes.

Six-valent osmium (osmate) complexes with nitrogenous ligands have previously been used for the modification and redox labeling of biomolecules involving vicinal diol moieties (typically, saccharides or RNA). In this work, aliphatic (3,4-dihydroxybutyl and 3,4-dihydroxybut-1-ynyl) or cyclic (6-oxo-6-(cis-3,4-dihydroxypyrrolidin-1-yl)hex-2-yn-1-yl, PDI) vicinal diols are attached to nucleobases to functionalize DNA for subsequent redox labeling with osmium(VI) complexes. The diol-linked 2'-deoxyribonucleoside triphosphates were used for the polymerase synthesis of diol-linked DNA, which, upon treatment with K2 OsO3 and bidentate nitrogen ligands, gave the desired Os-labeled DNA, which were characterized by means of the gel-shift assay and ESI-MS. Through ex situ square-wave voltammetry at a basal plane pyrolytic graphite electrode, the efficiency of modification/labeling of individual diols was evaluated. The results show that the cyclic cis-diol (PDI) was a better target for osmylation than that of the flexible aliphatic ones (alkyl- or alkynyl-linked). The osmate adduct-specific voltammetric signal obtained for OsVI -treated DNA decorated with PDI showed good proportionality to the number of PDI per DNA molecule. The OsVI reagents (unlike OsO4 ) do not attack nucleobases; thus offering specificity of modification on the introduced glycol targets.

Distinguishing the glycan isomers 2,3-sialyllactose and 2,6-sialyllactose by voltammetry after modification with osmium(VI) complexes.

Altered glycosylation is a universal feature of cancer cells and certain glycans are well-known markers of tumor progression. In this work we studied two glycan isomers, 2,3-sialyllactose (3-SL) and 2,6-sialyllactose (6-SL), frequently appearing in glycoproteins connected with cancer. A combination of square wave voltammetry and glycan modification with osmium(VI) N,N,N',N'-tetramethylethylenediamine (Os(VI)tem) allowed to distinguish between these regioisomers, since the 6-SL molecule can bind three Os(VI), while the 3-SL only two Os(VI) moieties, as experiments using capillary electrophoresis, inductively coupled plasma mass spectrometry and thin layer chromatography showed. A similar pattern of Os(VI)-modification was found for isomers of sialyl-N-acetyllactosamine and sialylgalactose. Covalent adducts of Os(VI)tem with glycans yielded three reduction voltammetric peaks. The ratio of peak I/peak II heights depends on the content of individual regioisomer in the sample. Our proposed approach allows the determination of isomer percentage representation in the mixture after one voltammogram recording. These results show a new appropriate method for the discrimination of glycan isomers containing terminal sialic acid important for distinguishing between cancerous and non-cancerous origin of biomarkers.

Immunoassays of chemically modified polysaccharides, glycans in glycoproteins and ribose in nucleic acids.

Glycosylation of proteins plays an important role in health and diseases. At present new simple and inexpensive methods of glycoprotein analysis are sought. We developed a monoclonal antibody Manost 2.1 in mice after immunization with the adduct of mannan with Os(VI)temed complex (temed is N,N,N',N'-tetramethylethylenediamine). The specificity of this antibody to different biomolecules treated with Os(VI)temed was tested using dot blot immunoassay. Manost 2.1 showed specificity toward Os(VI)temed-modified polysaccharides, glycoproteins and ribonucleotide at the 3'-end in DNA. The antibody recognized neither the unmodified compounds nor the non-glycosylated proteins treated with Os(VI)temed. We also performed western blotting and Coomassie silver blue staining of mixtures of biomacromolecules treated with Os(VI)temed and identified specifically the modified glycoproteins. The immunochemical method using Manost 2.1 was compared with electrochemical analyses based on redox signals of the Os(VI)temed adducts, with similar results in terms of sensitivity. This new antibody-based approach opens the door for rapid and inexpensive analysis of glycans and glycoproteins in various scientific and medical fields, including cancer research and the future application of glycoprotein detection in clinical practice.

Voltammetric determination of Os(VI)-modified oligosaccharides at nanomolar level.

Glycoproteins participate in various biological events, including disease progression. Currently, there is a pressing need for development of new simple and inexpensive methods for glycoprotein carbohydrate component (mostly oligosaccharides, OLSs) analysis and electrochemical methods were little applied in their analysis. Polysaccharides and OLS were long time considered as electroinactive compounds. We show that OLS adducts with six-valent osmium complexes are electroactive and can be determined at mercury and carbon electrodes. Adducts of OLSs with complex of Os(VI) with N,N,N',N'-tetramethylethylenediamine (tmen) can be prepared by mixing of OLS with [Os(VI)tmen] either at 37°C overnight or at 75°C in 10-15min. We modified 3α,6α-mannopentaose (MPO), stachyose and γ-cyclodextrin with [Os(VI)tmen]. The OLS adducts produced CV redox couples at hanging mercury drop electrode (HMDE) and at pyrolytic graphite electrode (PGE). 6nM MPO was determined by conventional adsorptive stripping at HMDE with RSD 5.3% directly in the reaction mixture. Similar determination at PGE was much less sensitive. Using adsorptive transfer (ex situ) stripping at PGE, µL volumes of OLS were sufficient for the analysis. Protein glycosylation stands at present in focus of medicinal chemistry because of its importance in various diseases and their diagnostics. Our paper represents first steps toward application of electrochemical methods in biomedical analysis of OLS.

Electrocatalysis in proteins, nucleic acids and carbohydrates.

The ability of proteins to catalyze hydrogen evolution has been known for more than 80 years, but the poorly developed d.c. polarographic "pre-sodium wave" was of little analytical use. Recently, we have shown that by using constant current chronopotentiometric stripping analysis, proteins produce a well-developed peak H at hanging mercury drop and solid amalgam electrodes. Peak H sensitively reflects changes in protein structures due to protein denaturation, single amino acid exchange, etc. at the picomole level. Unmodified DNA and RNA do not yield such a peak, but they produce electrocatalytic voltammetric signals after modification with osmium tetroxide complexes with nitrogen ligands [Os(VIII)L], binding covalently to pyrimidine bases in nucleic acids. Recently, it has been shown that six-valent [Os(VI)L] complexes bind to 1,2-diols in polysaccharides and oligosaccharides, producing voltammetric responses similar to those of DNA-Os(VIII)L adducts. Electrocatalytic peaks produced by Os-modified nucleic acids, proteins (reaction with tryptophan residues) and carbohydrates are due to the catalytic hydrogen evolution, allowing determination of oligomers at the picomolar level.

Electrocatalytic detection of polysaccharides at picomolar concentrations.

Electroinactive polysaccharides (PS) modified by osmium(VI) complexes with nitrogenous ligands produce redox couples at carbon and mercury electrodes. We show that PS adducts with Os(VI) 2,2'-bipyridine produce at ~-1.2 V (against Ag/AgCl/3 M KCl electrode) an additional peak at mercury and solid amalgam electrodes. This peak is due to the catalytic hydrogen evolution, allowing detection of PS (such as dextran and mannan) at picomolar concentrations.

"Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes.

Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.