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Analysis of the hydrolysis of inulin using real time 1H NMR spectroscopy.

ABSTRACT: The hydrolysis of various carbohydrates was investigated under acidic conditions in real time by (1)H NMR spectroscopy, with a focus on the polysaccharide inulin. Sucrose was used as a model compound to illustrate the applicability of this technique. The hydrolysis of sucrose was shown to follow pseudo first order kinetics and have an activation energy of 107.0 kJ mol(-1) (SD 1.7 kJ mol(-1)). Inulin, pullulan and glycogen also all followed pseudo first order kinetics, but had an initiation phase at least partially generated by the protonation of the glycosidic bonds. It was also demonstrated that polysaccharide chain length has an effect on the hydrolysis of inulin. For short chain inulin (DPn 18, SD 0.70) the activation energy calculated for the hydrolytic cleavage of glucose was similar to sucrose at 108.5 kJ mol(-1) (SD 0.60). For long chain inulin (DPn 30, SD 1.3) the activation energy for the hydrolytic cleavage of glucose was reduced to 80.5 kJ mol(-1) (SD 2.3 kJ mol(-1)). This anomaly has been attributed to varied conformations for the two different lengths of inulin chain in solution.

SUBMITTER: Barclay T

PROVIDER: S-EPMC3324600 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Analysis of the hydrolysis of inulin using real time 1H NMR spectroscopy.

Barclay Thomas T Ginic-Markovic Milena M Johnston Martin R MR Cooper Peter D PD Petrovsky Nikolai N

Carbohydrate research 20120310

The hydrolysis of various carbohydrates was investigated under acidic conditions in real time by (1)H NMR spectroscopy, with a focus on the polysaccharide inulin. Sucrose was used as a model compound to illustrate the applicability of this technique. The hydrolysis of sucrose was shown to follow pseudo first order kinetics and have an activation energy of 107.0 kJ mol(-1) (SD 1.7 kJ mol(-1)). Inulin, pullulan and glycogen also all followed pseudo first order kinetics, but had an initiation phase ...[more]

PMID: 22464225

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Project description:APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3'→5' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5'-end is deaminated more effectively than one less close to the 5'-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2's activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3'→5' polarity. Examination of the effects of different salt concentrations on the 3'→5' polarity indicated that the higher the salt concentration, the less prominent the 3'→5' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.

Project description:BackgroundAmbiguity in malignant transformation of glioma has made prognostic diagnosis very challenging. Tumor malignant transformation is closely correlated with specific alterations of the metabolic profile. Exploration of the underlying metabolic alterations in glioma cells of different malignant degree is therefore vital to develop metabolic biomarkers for prognosis monitoring.MethodsWe conducted (1)H nuclear magnetic resonance (NMR)-based metabolic analysis on cell lines (CHG5, SHG44, U87, U118, U251) developed from gliomas of different malignant grades (WHO II and WHO IV). Several methods were applied to analyze the (1)H-NMR spectral data of polar extracts of cell lines and to identify characteristic metabolites, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), fuzzy c-means clustering (FCM) analysis and orthogonal projection to latent structure with discriminant analysis (OPLS-DA). The expression analyses of glial fibrillary acidic protein (GFAP) and matrix metal proteinases (MMP-9) were used to assess malignant behaviors of cell lines. GeneGo pathway analysis was used to associate characteristic metabolites with malignant behavior protein markers GFAP and MMP-9.ResultsStable and distinct metabolic profiles of the five cell lines were obtained. The metabolic profiles of the low malignancy grade group (CHG5, SHG44) were clearly distinguished from those of the high malignancy grade group (U87, U118, U251). Seventeen characteristic metabolites were identified that could distinguish the metabolic profiles of the two groups, nine of which were mapped to processes related to GFAP and MMP-9. Furthermore, the results from both quantitative comparison and metabolic correlation analysis indicated that the significantly altered metabolites were primarily involved in perturbation of metabolic pathways of tricarboxylic acid (TCA) cycle anaplerotic flux, amino acid metabolism, anti-oxidant mechanism and choline metabolism, which could be correlated with the changes in the glioma cells' malignant behaviors.ConclusionsOur results reveal the metabolic heterogeneity of glioma cell lines with different degrees of malignancy. The obtained metabolic profiles and characteristic metabolites are closely associated with the malignant features of glioma cells, which may lay the basis for both determining the molecular mechanisms underlying glioma malignant transformation and exploiting non-invasive biomarkers for prognosis monitoring.

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Analysis of the hydrolysis of inulin using real time 1H NMR spectroscopy.

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Analysis of the hydrolysis of inulin using real time 1H NMR spectroscopy.

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