Tritiation sugar

Immune rabbit serum, 5 yl is mixed with varying amounts of inhibitor in buffer in a final volume of 300 pi. After incubation for 2 h at 25°, 6.7 pinole of [ 3H ]mannotetraose (103 cpm) in 100 pi of buffer is added and incubation is continued for 2 h at 25°. Binding of tritiated sugar is determined as described in Figure 2. 

It is very important to perform this reaction uncapped in a fume hood to prevent the room from filling with tritium gas. The mixture of tritiated sugar alcohols is then separated by paper chromatography on thin strips. These strips are divided into half-inch segments and analyzed by liquid scintillation counting. The total counts per minute of 3H in each sugar alcohol peak can then be used to calculate the amount of each carbohydrate present in the sample. 

Most tritiated sugars are best stored in aqueous solution, sometimes containing a small proportion of ethanol. Under these conditions, radiolysis usually does not exceed 5 to 10% per year. In the frozen state, between 0° and —196°, the rate of decomposition can be high, presumably due to concentration of the sugar in pockets on freezing.87 Nucleotides are stored at —20° in 50% ethanol, which acts as a free-radical scavenger,68 and decompose at the rate of 12% per year polymers should be stored as freeze-dried solids. 

The relative ease of synthesis of tritiated sugars, as compared with that of 14C-labeled sugars, has led to the increasing use of tritiated substrates in the determination of the molecular course of a particular biological transformation. These studies, although limited... 

Glycolipids moving along an identified axon of the marine mollusc, Aplysia californica, were synthesized and incorporated into intracytoplasmic membranes solely in the perikarion. Direct injection of tritiated sugar into the axon revealed no localized synthesis or exchange. There was no indication of any transfer into the axoplasm from the glia. The insertions of glycolipids into nascent membranes occurs co-ordinately with the insertion of protein components in the cell body. 

Reduction with tritiated NaBH4 (NaB3H4), followed by analysis of the radioactive sugar alcohols by paper chromatography and liquid scintillation counting. 

Non-enzymic aldose-ketose isomerisations that are acid catalysed appear to involve a 1,2-hydride shift. During acid-catalysed rearrangement of glucose to fructose, the label of [2- H]glucose substrate is retained in the [l- H]fructose product, distributed equally between the proR and proS positions." In the reverse sense retention of the label of tritiated fructose in the glucose and mannose products was not complete. Similar observations were made for the xylose-xylulose interconversion." With an appropriate sugar configuration (ribose), even the base-catalysed reaction proceeds partly with retention of label, presumably by the same mechanism as with trioses. 

Nucleosides prepared conventionally from deoxy-sugars have included the antiviral nucleoside dihydro-5-azathymidine (12), the tritiated analogue (13), and the 6-substituted derivatives (14) (Scheme 2), [ C-2] and [ C-4]-2-deoxy-uridine, and 2-deoxy-ribonucleosides from 5-azapyrimidine derivatives, ... 

Applications of h.p.l.c. to benzoyl derivatives of alditols, glycosides, amino-sugars, aldoses, and alditols has been examined the presence of benzoyl groups has the advantage that u.v. detection methods can be used, and indeed a method for the simultaneous determination of picomole levels of gluco- and galacto-cerebrosides involves benzoylation followed by h.p.l.c.Similarly, tritiated mannitol and fucitol were easily separated after O-benzoylation. ... 

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