Radioiodine labeling, protein

Denaturation of proteins may result from low concentrations and elevated storage temperatures. These conditions must also be controlled for radioiodinated proteins that are also subject to internal self-irradiation damage, which is more pronounced in I-labeled proteins than I-la-beled proteins owing to the beta radiation emission of I. To minimize such problems, radioiodinated proteins are diluted in protective protein solutions that will not interfere in the subsequent studies to be made, such as 10% normal serum or BSA at 2 mg of protein per milliliter. Aliquots are kept frozen at -20° to -70° and thawed only as needed. 

Figure 1 shows the kinetics of release of radioiodinated protein from antibody-complement-treated tumor cells. Guinea pig serum caused the maximum enhanced release compared to untreated cells of I-labeled protein from anti-Forssman antibody-sensitized cells within 10 min of incubation there was no enhanced release of I-labeled protein compared to controls from anti-line-10 antibody-sensitized cells treated with GPC (Fig. lA). Similarly, HuC caused maximal enhanced release of cell surface protein from cells sensitized with either antibody within 10 min (Fig. IE). No enhanced release of membrane protein was observed from cells treated with antibody alone or complement alone (Fig. 1). The values in Fig. 1 represent 23-40% of the total cell-bound activity associated with [ I]ISA.

Under these conditions, radioiodine incorporation of up to 99% can be achieved at labeled protein recovery rates of >90%. The reaction is more gentle than soluble Chloramine-T because there is less contact between the protein and the immobilized oxidizing agent. If the reaction is conducted at pH values above 8, it is possible to radioiodinate histidine residues. The reaction is believed to proceed in the close proximity of the surface of the polymer beads. An electrophilic iodosulfonamide intermediate species that reacts with either tyrosine or histidine residues of the protein to be labeled is formed. 

In search of alternatives to Chloramine-T, Morrison (1980) demonstrated that lactoperoxidase from bovine milk was particularly effective for iodide oxidation and the radioiodination of proteins. The single strand polypeptide lactoperoxidase has a molecular weight of approximately 78 kD. The structure of lactoperoxidase contains a prosthetic heme group which is covalently linked to the protein at the active site of the enzyme. In the presence of minute quantities of hydrogen peroxide, lactoperoxidase oxidizes and binds radioiodide to proteins in the reaction mixture. The pH optimum of iodide oxidation by lactoperoxidase is 4-8.5 with an optimum at pH 5. The reaction is extremely rapid, allowing reaction times of less than 1 min. This provides very mild conditions and denaturation of proteins is low. Yields of up to 85% can be obtained and the method is widely used for labeling proteins and hormones. Lactoperoxidase may itself... 

Chattopadhyay et al., 1992), and a comparison of radiolabeling techniques for the crosslinker (Shephard et al., 1988). Other studies have involved the investigation of protein interactions using the label transfer nature of radioiodinated SASD (Gupta et al., 2005 Lindersson et al., 2005 LeFebvre et al., 2006). 

For the selective labeling of the acetylcholine receptor Saitoh et al. used the covalently bound non-competitive blocker 5-azido- H-trimethioquin. Asymmetric labeling of the proteins in the kidney microvillar membrane by lactoperoxidase-catalysed radioiodination and by photolysis of 3,5-di- I-4-azidobenzenesulphonate is a new approach which may be applied to the topological investigation of ojmplex membranes... 

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