Rapid Genotype Method to Distinguish Frequent and/or Functional Polymorphisms in Human N-Acetyltransferase-1
✍ Scribed by Mark A. Doll; David W. Hein
- Book ID
- 102562169
- Publisher
- Elsevier Science
- Year
- 2002
- Tongue
- English
- Weight
- 63 KB
- Volume
- 301
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
ppm), due to the  effect. The same effect for compound 4 shifts the C-6 signal, which is observed at 66.2 ppm. Deprotection of HO-6 also affects the pattern of H-6,6Ј in the 1 H NMR spectrum of 3. In this case, the two protons appear as a doublet at the same chemical shift (4.07 ppm, Table 1).
Oxidation of HO-6 was performed with PCC as described (12) affording 5 (88%). Compound 5 showed a singlet at 9.2 ppm in the 1 H NMR spectrum, and a signal at ␦ 196.5 in the 13 C NMR spectrum, both diag- nostic signals for the presence of the aldehyde group at C-6. For the reduction and debenzoylation steps, standard procedures were used. Compound [ 3 H]1 showed the same chromatographic properties by TLC (Fig. 1, lane I) and HPAEC-PAD (Fig. 2A) than the nonradioactive compound. The specific activity (13 Ci/mol) was calculated from the ratio between the radioactivity of the compound eluted from the HPAEC and the amount of material calculated by using mannose as standard.
Compound [ 3 H]1 was used as substrate for the exo--D-galactofuranosidase from P. fellutanum, under the usual conditions ( 6). The enzymatic reaction could be followed by TLC (Fig. 1, lane II) and HPAEC-PAD analysis (Fig. 2B). This is the first report on the synthesis of a radioactive substrate for -D-galactofuranosidase. The radiolabeled material will facilitate detection of the enzyme in cultures or cellular fractions of microorganisms. In addition to being more sensitive, it can be used with colored biological materials without the interference caused in the colorimetric assay. The method could be useful for labeling other substrates in studies on the biosynthesis of galactofuranose glycans, like the galactan of Mycobacterium tuberculosis (13), although this chemical-labeling procedure would not be specific when other monosaccharides are present.
Acknowledgments.