Double-labelled molecules

Phthalic acid anhydride reacts readily with alcoholic end groups of PEG. Besides an appropriate chromophore, charges are introduced simultaneously. Single- and double-labeled molecules are formed when two possible alcohohc end groups are available. The separation of derivatives of low molecular weight, such as PEG 1000, is shown in Fig. 14. Here, no sieving matrix was added. The homo-logues of the double labeled derivatives were completely separated [32]. 

In considering the ring-closure of (7.53) to give (7.54) and the apparently connected double-bond isomerization, knowledge of the fate of the proton at C-9 in (7.53) is important. It was found to be incompletely retained ( 70%) during biosynthesis (that at C-10 is completely retained). In an experiment with a 1 1 mixture of [2- C]-and [4- H2]-mevalonate, the derived (7.53) showed the expected mixture of singly labelled species, but the elymoclavine (7.55) showed an appreciable percentage of double-labelled molecules, i.e. molecules which contained both C and deuterium. These results and the required double-bond isomerization are neatly accounted for in terms of the mechanism shown in Scheme 7.6 [30] in which there is an... 

In another experiment, [l,2- C2-2-dJ double-labeled acetate was fed. First we observed a complete loss of deuterium atoms. In a short incubation, however, we obtained neosaxitoxin partially retaining a deuterium atom (40% equivalent of incorporated acetate molecule). The location of the deuterium atom was on C-5, which was originally the carboxyl carbon of acetate, suggesting that it migrated from the adjacent methyl-derived carbon C-6. 

Rotational-echo double-resonance (REDOR)(75,79) is a new solid-state NMR technique which is sensitive to through-space carbon-nitrogen interactions between selectively 13C and 15N-enriched sites separated by up to 5A (20-22). The parameter directly measured in a REDOR experiment is the heteronuclear dipolar coupling constant DCN, which is in itself proportional to the inverse third power of the intemuclear distance, rCN. It is this dependence on (icn)3 which accounts both for REDOR s ability to accurately measure short distances and its insensitivity to longer-range interactions. As a technique which can probe, in detail, intermolecular interactions over a distance range of 5A, REDOR is well suited to studying the distribution of small selectively-labeled molecules in polymer delivery systems. 

In a similar fashion, steroids are molecules that have been investigated by disruption of FRET. The sensor is a double labeled peptide with cyclodextrin bound to one side chain. The latter keeps the fluorophores closely together by accommodating the coumarin into its cavity thereby ensuring efficient FRET. Steroids compete for the cavity of cyclodextrin and displace the coumarin reducing FRET efficiency. This model, although useful for in vitro applications, seems to be poorly selective for its application in biological samples [95],... 

Remote double labelling techniques have been used successfully in the determination of enzyme KIEs (Kiick, 1991). A variant of this technique was applied to a nonenzymatic reaction by Matsson and co-workers (Axelsson et al., 1990). They determined the primary carbon and secondary deuterium KIEs for the SN2 reaction between methyl iodide and hydroxide ion in 50% dioxane-water at 25°C. The a-carbon KIE was determined by the UC method (Axelsson et al. 1987,1991). In this method, a mixture of substrate molecules labelled with UC (tm = 20.4 min) and 14C is used. The reactants and products... 

The position of the label should be away from sites chemically instable or from sites of metabolic attack to ensure that the label is kept in the main metabolic fragments. Of course, this is difficult for complex molecules, especially when the metabolic attack takes part in the center of the drag molecule. Then it can become necessary to introduce a second label and to repeat the set of radiokinetic studies. A double-labeling strategy to minimize the number of radiokinetic studies has normally to be refused due to complexity of interpretation of the data. 

In fact, no singly labelled compounds were found NMR analysis showed that the product consisted entirely of unlabelled or doubly labelled molecules. The CO2H group remains attached to the same molecule (though not to the same atom) and the first mechanism is correct. Crossover experiments demand some sort of double labelling, which does not have to be isotopic. An example where crossover products are observed is the light-initiated isomerization of allylic sulfides. 

The position of labelling must be chosen in such a way that the products of metabolism can be identified, e.g. by labelling with T or " C in the side chain of an aromatic compound if the fate of the side chain is of interest, or labelling in the aromatic part of the molecule if this is the main object of investigation. Double labelling, for instance by T and " C at different positions, is often very helpful. 

The basic concepts of competitive-binding solid phase EIA have been described elsewhere.The required separation of antibody from the assay mixture can be accomplished in a variety of ways. Double-antibody techniques are quite popular and involve the use of a second antibody, an antibody to the principal antibody, to induce separation. Cross-linking the second antibody serum - with ethyl chloroformate forms an insoluble suspension of small particles, which still maintain a high degree of im-munoreactivity toward the first antibody. Addition of such particles to an EIA assay mixture pulls the first antibody from solution along with enzyme-labeled molecules bound to the antibody. Such a process is depicted in Fig. 1 and employed for all separations in this work. Measurement of enzyme activity in the bound solid phase is desirable because complete purification of the labeled substance is not necessary. 

Recombinant human leptin was recently cloned (8) and expressed in E. coli, and demonstrated to effectively regulate adiposity in mice through modulation of appetite and metabolism (9, 10). The molecule contains four methionine residues at positions 1, 54, 68, and 136. In this paper, we report the separation and characterization of three norleucine-incorporated recombinant human leptins which were uniformly labeled with 15n isotope or double labeled with and isotopes. The extent of incorporation at each methionine residue can be determined by reverse-phase HPLC and amino acid analysis methods. The norleucine incorporation was observed preferentially occurring at the internal Met residues. 

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