Oxygen labeling requirements

The oxygen formed clearly comes from H20 and not from C02, because photosynthesis in the presence of water labeled with lgO produces oxygen labeled with 180, whereas carbon dioxide labeled with 180 does not give oxygen labeled with 180. Notice that the oxidation of the water produces two electrons, and that the formation of NADPH from NADP requires two electrons. These reactions occur at different locations within the chloroplasts and in the process of transferring electrons from the water oxidation site to the NADP reduction site, adenosine diphosphate (ADP) is converted to adenosine triphosphate (ATP see Section 15-5F for discussion of the importance of such phosphorylations). Thus electron transport between the two photoprocesses is coupled to phosphorylation. This process is called photophosphorylation (Figure 20-7). 

Figure 8.4 Conversion of acyl-CoAs to hydrocarbon in the housefly. Appoximately equal amounts of labeled hydrocarbon and carbon dioxide are produced from the microsomal conversion of [1-14C, 15,16-3H]24 1-CoA to products (A). The inset shows the retention times of labeled CO and C02 (A, B) and product from [1-14C]24 1-CoA (AA). NADPH and oxygen are required for microsomal conversion of 24 -CoA to hydrocarbon (B) and (C) shows the steps involved in hydrocarbon formation (data from Reed et at, 1994).

Special Labeling Requirements for Etiologic ents. Oxygen, Chlorine, and Radioactive Materials Special labeling requirements exist for etiologic agents, oxygen, chlorine, and radioactive materials, certain of which operate to impose additional requirements while others establish exceptions from otherwise applicable requirements. 

Bilirubin and carbon monoxide are always produced in equimolar proportions under normal conditions of heme catabolism the process is oxidative and requires molecular oxygen. Labelling studies using 02 show that the labelled oxygen ends up in the bilirubin at the terminal lactam positions, and in the carbon monoxide that is liberated (by removal of the C-5 carbon). The overall process is shown in Figure 5.5. 

An unusual monounsaturated fatty acid, specifically linked to phosphatidyl glycerol and found in chloroplasts, is rran -S-hexadecenoic acid (Table 3.2). Palmitic acid is its precursor and oxygen is required as cofactor. When labelled ran5-3-hexadecenoic acid is incubated with chloroplast preparations, it is not specifically esterified in phosphatidylglycerol but is either randomly esterified in all chloroplast lipids or reduced to palmitic acid. These results could be explained if the direct precursor of rrflw -3-hexadecenoic acid were palmitoyl-phosphatidylglycerol and not palmitoyl-5-CoA or palmitoyl-5-ACP. However, the precise details of the reaction have yet to be worked out. 

Peracetic acid specifically labelled with oxygen-18 has been used for the oxidation of (+ )-benzyl-p-tolyl sulphoxide to the (— )-[160180]sulphone46 (equation 15). The reaction gives the required enantiomer in no less than 80% yield. 

It may be noted that 16 are stable compounds hence it should be possible to prepare them independently and to subject them to the conditions of the von Richter rearrangement. This was done and the correct products are obtained. Further evidence is that when 15 (Z=C1 or Br) was treated with cyanide in H O, one-half the oxygen in the product was labeled, showing that one of the oxygens of the carboxyl group came from the nitro group and one from the solvent, as required by this mechanism. ... 

The metabolites and metabolic pathway of a new anticonvulsant drug, sodium valproate, in rats were investigated using carbon-14 labeled sodium valproate. Most of the metabolites in urine and bile were a glucuronide conjugate of valproic acid. Free sodium valproate was as little as one-seventh of the total metabolites. In feces, only free sodium valproate was detected, and the possibility of enterohepatic circulation of sodium valproate was presumed. A part of dosed sodium valproate was excreted in expired air in the form of CO2. This degradative reaction took place in liter mitochondria and required CoA and oxygen. It was stimulated by ATP... 

Comparison of the results allows calculation of kj6/ki8. Obviously there are drawbacks to this procedure. The major one is the necessity of a costly and tedious isotopic synthesis of labeled materials. Optimally those compounds should be as close as possible to 100% enriched. This can seldom be achieved and using partially enriched samples requires substantial corrections to the raw data and increases experimental uncertainty. A rule of thumb used in remote labeling experiments is that the remote (reporting) position should be reasonably far from the reaction center (the phenolic oxygen in the present example). For the case where there is no isotope effect at the reporting site (e.g. no 15N-KIE), the double-label experiment leads directly to the isotope effect of interest. This is more probable when the reporting site is remote, (i.e. well isolated from the reaction coordinate). 

An O-labelling investigation of the oxygen to sulfur transposition in the base-catalysed rearrangement of o-benzoyl-A-(diphenylphosphinothioyl)hydroxylamine (200) to (201) has been undertaken. The labelling results are outlined in Scheme 70 although further evidence is required to substantiate the mechanism... 

This step in the reaction cycle requires one O2 molecule and two electrons to give Fe + biliverdin with a third electron needed to reduce Fe + to Fe + followed by release of the iron from biliverdin 176, 188, 198,199). A hydrolytic mechanism for the incorporation of oxygen in the conversion of verdoheme to biliverdin is ruled out because O labeling experiments show incorporation of into biliverdin 200). Details on the mechanism remain sketchy, and one possible pathway is illustrated in Fig. 21, taken after Ref 176). 

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