Kilian Dill

Detection of salmonella in poultry using a silicon chip-based biosensor.

Salmonella typhimurium was detected to levels as low as 119 CFUs using the Threshold Immunoassay System. This immunoassay system utilizes solution-based binding of the biotin and fluorescein labeled antibodies to salmonella, followed by filtration-capture of the immunocomplex on a biotin-coated nitrocellulose membrane. Lastly, an anti-fluorescein urease conjugate is bound to the immunocomplex. Detection of the bound immunocomplex is made possible via the silicon chip-based light-addressable potentiometric sensor. In the presence of the urea, urease converts the substrate to ammonia and CO2 and this results in a pH change at the silicon surface. The resultant pH change is monitored with time and the signal output is reported in microV s(-1). An experiment whereby chicken carcass washings were fortified with salmonella showed a recovery of 90%, indicating that the technique can be used to test for salmonella under these conditions. Precautions must be used with this instrument as sample debris will affect sample flow through the membrane and hence the signal output.

Natural-Abundance, 13C-Nuclear Magnetic Resonance-Spectral Studies of Carbohydrates Linked to Amino Acids and Proteins

Publisher Summary This chapter presents the 13 C-nuclear magnetic resonance-spectral studies of carbohydrates linked to amino acids and proteins. This technique is dealt with in terms of the information that may be gained about the glycoprotein without its modification, which would lead to the destruction of the native sample and possible microheterogeneity of the oligosaccharide chains. The covalent attachment of carbohydrates to amino acid residues of glycoproteins falls into three main categories including the glycosidic ( O -glycosyl) linkage, the N -glycosyl linkage, and the S -glycosyl linkage. The analysis of the anomeric-carbon region of two glycoproteins— human α 1 -acid glycoprotein (α 1 -AGP) and calf fetuin—provides additional information not available by more conventional methods. Any structural analysis of such a complex system must be based on the assignments of the various signals in the spectra. Their spectra shows that resonances corresponding to inner carbohydrate display residues a much larger line-width than resonances corresponding to outer residues. This reflects the relative mobility of such residues and aids in distinguishing between terminal residues and inner residues of the intact oligosaccharide chains of intact glycoproteins. The structural analysis of glycopeptides and glycoprotein containing O -glycosyl linkages is also elaborated in the chapter.

13C-N.M.R.-Spectral study of the mode of binding of Mn2+ and Gd3+ to N-acetyl-α-neuraminic acid

Abstract Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd3+ and Mn2+ to N-acetyl-2-O-methyl-α-neuraminic acid (2) and to methyl N-acetyl-2-O-methyl-α-neuraminate (3). The results showed that Gd3+ and Mn2+ bind in the region of the glycerol-1-yl side-chain and the 5-acetamido group of compound 3. When the α-NeuAc derivative contains a carboxylate anion, as in compound 2, multiple, metal-ion-binding sites occur, involving the head (the carboxyl end) and the tail (the glycerol-1-yl and 5-acetamido groups) of the molecule.

13C-n.m.r.-spectral study of the binding of Gd3+ to glycophorin

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd3+ to glycophorin, and also to the tetrasaccharides isolated from glycophorin after treatment of the glycoprotein with NaOH-NaBH4. Gd3+ binds to the tetrasaccharide (both in the isolated, reduced form and when still attached to the native glycoprotein), and, especially, to the alpha-NeuAc residues. In order to cause severe line-broadening of the 13C resonances of alpha-NeuAc, the ratios of the alpha-NeuAc residues of glycophorin, and of the isolated, reduced tetrasaccharide, to Gd3+ were much higher than that needed for causing similar broadening for 2-O-methyl-alpha-NeuAc-Gd3+ solutions. These results indicate that the other carbohydrate residues of the tetrasaccharide may be involved in the binding of Gd3+, producing a stronger metal-ion-binding effect.

One-dimensional and two-dimensional nuclear magnetic resonance studies of the reaction of phenyldichloroarsine with glutathione

14C-labeled phenyldichloroarsine (PDA) enters the red blood cell and forms a 1:2 adduct with intracellular glutathione. Upon gel filtration of the hemolysate, [14C]PDA was recovered with the glutathione-containing fractions. One-dimensional and two-dimensional nuclear magnetic resonance spectroscopy were used to confirm the structure of the adduct and elucidate its stereochemistry, stability, and reactivity.

Specific 13C reductive methylation of glycophorin A. Possible relation of the N-terminal amino acid and the lysine residues to MN blood group specificities

Heterozygous and homozygous glycophorin A were partially and fully reductively methylated with 13C-enriched formaldehyde in the presence of sodium cyanoborohydride. Total reductive methylation modified the five lysine residues (to produce N epsilon,N-[13C]dimethyl lysine) and the N-terminal amino acid residues (N alpha,N-[13C]dimethyl serine and leucine) of glycophorins AM and AN, respectively. 13C-NMR spectra of these species indicated that the 13C-enriched methyl carbons of the five lysyl derivatives all occur at 44.1 ppm downfield from Me4Si. Titration results indicate that the pK alpha of these methylated lysines is greater than 10. The chemical shift equivalent methyl resonances of the 13C-enriched methylated N-terminal Leu derivative were found to occur at 42.8 ppm downfield from Me4Si and exhibited a normal pH titration behavior (pK alpha approximately 7.4). The methyl resonances of the N alpha,N-[13C]dimethyl Ser derivative, on the other hand, were found to exhibit chemical shift nonequivalence, indicating rotational constraints about the C alpha-N bond. The linewidths of the two methyl resonances were also found to be considerably different; this phenomenon could be eliminated by running spectra of the sample (pH approximately 5.0) at elevated temperatures (75 degrees C). This result suggested that for the N alpha,N-[13C]dimethyl Ser derivative of glycophorin AM, hindered rotation must occur about one of the N alpha-13CH3 bonds. This structural difference at the N-terminal residue of glycophorins AM and AN may be related to the MN blood group determinants displayed by these related glycoproteins.

Possible role of the carbohydrate residues in the display of the MN blood group determinants by glycophorina

Heterozygous glycophorin AM,N and homozygous glycophorin AM were reductively methylated with 13C-enriched formaldehyde in the presence of cyanoborohydride. Total reductive methylation modified the five lysine residues, and the N-terminal amino acid residues (serine and leucine) of glycophorins AM and AN, respectively. The 13C resonances of the incorporated labels were monitored as a function of the degree of glycosylation of the glycoprotein. While minimal, if any, structural changes were observed near the N-terminal amino acid upon removal of alpha-D-N-acetylneuraminic acid residues, gross structural changes were observed when most of the oligosaccharide chains were removed. We also found that progressive methylation of the lysine residues of glycophorin AM may influence either the chemical shift of one of the nonequivalent methyl groups of the N alpha, N-[13C]dimethyl serine residue, or one of the two states of glycophorin AM.

Magnetic resonance study of glycophorin A-containing 13C-enriched methionines

Methionine‐81 and/or ‐8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with 13CH3I to produce the sulfonium ion derivatives [S‐[13C]methylmethionine‐8]glycophorin A and [S‐[13C]methylmethionine‐8 and ‐81]glycophorin A. 13C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me4Si. A spin‐lattice relaxation time of 0.4 was observed for the 13C‐enriched methyl resonances of the sulfonium ion derivatives of Met‐8 and ‐81, which corresponds to an effective correlation time of < 2× 10−10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2‐mercaptoethanol produces native glycophorin A which now has the ε‐carbon of the methionine residue(s) 45% isotopically enriched. The ε‐carbon of Met‐8 was found to occur at 15.7 ppm downfield from Me4Si whereas the ε‐carbon of Met‐81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me4Si. The spin‐lattice relaxation time of both resonances was found to be ∼0.3 s.

13C-N.M.R.-spectral study of some mono- and di-O-galactosylated dipeptides. Possible structural perturbations due to O-glycosylation

Abstract Carbon-13 nuclear magnetic resonance data for mono- and di- O -α- and -β- D -galactosylated dipeptides composed of Thr and Gly are presented. The results conclusively show that peptide-bond formation does not affect the chemical shifts of the attached carbohydrate carbon atoms. In the case of the di- O -glycosylated threonyl-threonine, no carbohydrate-carbohydrate interactions could be observed. For some of the mono- O -glycosylated dipeptides, the attached glycosyl group appears to have a peculiar effect on the chemical shifts of some of the carbon resonances of the amino acids.

13C-Nuclear Magnetic Resonance-Spectral Studies of the Interactions of Metal Ions with Carbohydrates: use of Relaxation Probes

Publisher Summary This chapter elaborates the technique of 13 C-nuclear magnetic resonance (NMR) spectroscopy that has been used to study the binding of the metal ions to various carbohydrate residues and glycopeptides and it discusses the ways to extract information on the specific binding sites of the carbohydrate residues to define further how and why metal ions interact with certain residues in biological systems. It had been shown that as few as three neutral oxygen atoms suffice to define a binding site for metal ions if oxygen atoms are in the correct steric arrangement. It might be interesting to compare the tendencies in bonding because the metal ions typically used to study binding to carbohydrates are from the first-row transition-metals and from the lanthanides. Because of their large numbers of unpaired electrons, Gd 3+ and Mn 2+ have been considered as relaxation agents to be used in magnetic resonance imaging (MRI). Inositols provide a good starting point for studying the binding of metal ions such as Gd 3+ and Mn 2+ to carbohydrates because each is unique, they are structurally related to carbohydrates, and they do not contain other functional groups (carboxyl, amino) that may also interact with the metal ions.