Iodination lactoperoxidase

Figure 12.6 The immobilized glucose oxidase/lactoperoxidase system radioiodinates proteins through the intermediate formation of hydrogen peroxide from the oxidation of glucose. H2O2 then reacts with iodide anions to form reactive iodine (I2). This efficiently drives the formation of the highly reactive H2OI+ species that is capable of iodinating tyrosine or histidine residues (see Figure 12.2).

Iodine becomes incorporated into proteins either oxidatively, or enzymatically, or electrochemically. Oxidative incorporation uses organochemical oxidants, such as, for example, chloramine T or lodo-gen (l,3,4,6-tetrachloro-3a,6a-diphenyl glycouril). Enzymatic incorporation is done by means of lactoperoxidase. By these methods iodine is introduced into phenyl (tyrosyl) residues of the protein. 

Isocratic RPC was also successful in separating the position isomers of mono-iodinated insulin. The 125-iodination by the lactoperoxidase method was carried out in phosphate buffer containing 6 M urea and led to the iodination of tyrosyl units, i.e., to substitution in the positions A14, A19, B16, or B26. The retention of the mono-iodinated insulin chains increased in the order A19 < B26 < B16 < A14 on a C 18 column (150 x 4.0 mm dP = 3 pm). The eluent contained 1 % triethylammonium tri-fluoroacetate in water (pH 3) and acetonitrile78). 

The first step in the biochemical analysis of the receptor, isolation from the cell membrane, was difficult the membrane-associated protein is present in only small amounts and is insoluble in aqueous solution. Sensitivity and selectivity would be improved by radiolabeling under conditions that maintain cell viability so that only proteins associated with the surface and not those within the cell become labeled. A procedure that met these conditions was iodination catalyzed by lactoperoxidase in the presence of hydrogen peroxide (Marchalonis, 1969 Phillips and Morrison, 1970 Marchalonis et al., 1971). It was also necessary to find conditions for solubilizing membrane proteins that would not interfere with subsequent specific immunoprecipitation. 

Cuatrecasas (45). The Dolichos biflorus lectin was a gift from Dr. Marilynn Etzler (46). Ulex europeus and Bandeirea simplicifolia lectins were purified in our laboratory and iodinated with Nal25i (carrier-free) by Sepharose 4B-bound lactoperoxidase (47)and diazotized iodoaniline coupling (48) procedures, respectively. 

The iodination with lactoperoxidase is also very gentle and very often used Because the application of an enzyme for oxidation is more expensive than the use of common oxidation reagents, procedures involving immobilized lactoperoxidase have been worked out 21.22.26)... 

There is considerable interest in reagents which covalently attach labels to cell surfaces. To be successful, intact cells and reagents too large to penetrate the cell surface must be used. To date most attention has been focused on the lactoperoxidase-catalyzed iodination of exposed tyrosine residues. The first work, done with human erythrocytes (181), has been extended to splenic lymphocytes (182). 

Two principal methods exist for incorporation of I or I into purified antigens. Both involve oxidizing iodide to active iodine, presumably a cationic form of iodine (f), which then reacts with ionized tyrosine residues of the antigen. The first method (12) employs lactoperoxidase and hydrogen peroxide (Figure 8-34) and the second method (14) uses... 

Fibrogen. Two methods were used to prepare I-labeled fibrinogen. In both methods, unincorporated iodide was removed immediately after the reaction by gel filtration on a G-25 Sephadex column preequilibrated in O.OIM HEPES, 0.147M NaCl, 0.02% azide, pH 7.4. The first method uses catalysis by lactoperoxidase and is based on studies by Marchalonis (23), and the second method uses iodine monochloride (ICI) and is based on work by Helmkamp et al. 24). 

The introduction of I into molecules containing tyrosyl or histidyl moieties can be carried out chemically by the Chloramine T procedure or enzymatically by lactoperoxidase." The prelabeled Bolton-Hunter reagent [A-succinimidyl 3-(4-hydroxyl-5-[ I]iodophenylpropionate] reacts under mild conditions with molecules containing amino groups (e.g., proteins and haptens) to introduce an iodinated propyltyrosyl moi-... 

Only two amino acids, tyrosine and histidine, form stable derivatives as the result of peroxidase-catalyzed iodination. All the tyrosine and histidine residues in a protein are not identical with respect to their reactivity or their geographic position. The residue which will be iodinated by lactoperoxidase must have the proper geometric position, while other methods of halogenation are influenced only by reactivity. The reactivity depends upon the microenvironment of the residue. There is an inverse relationship between the extent of tyrosine iodination and the dielectric constant of the environment of the tyrosine. Tyrosine iodination increases with decreasing dielectric constant. Steric factors also influence iodination since the relatively large iodine atom may be blocked in either the production of monoiodotyrosine or the formation of diiodotyrosine. 

We have employed a variety of procedures for removing free iodide from the iodinated proteins. This can be accomplished by (a) gel filtration, (b) dialysis, and (c) use of ion exchange resin. Gel filtration is a very effective method for removal of both iodide and lactoperoxidase if the molecular weight of the iodinated protein and the molecular weight of the enzyme are sufficiently different as in the example given. Since the iodine derivatives are light sensitive all procedures should be carried out in dim light. 

If gel filtration procedures cannot be employed for removal of lactoperoxidase, the enzyme can be removed by taking advantage of the fact that it is a basic protein. It is easily separated from most proteins which have lower isoelectric points by the use of ion exchange resin. The reaction mixture is passed through a small column of IRC 50 at a pH above the isoelectric point of iodinated protein but below pH 8.0. This will remove the lactoperoxidase from the mixture without adsorbing proteins of lower isoelectric points. 

In order to illustrate the use of the lactoperoxidase procedure as a vectorial probe, the example to be given is the iodination of the plasma membrane of the human lymphocyte. All manipulations were carried out at 4°. The lymphocytes were isolated and washed free of extracellular protein in isotonic phosphate buffer. The cells were centrifuged at 700 g for 10 min and the supernatant serum was removed. Cells are resuspended in 5 to 10 volumes of phosphate buffer and centrifuged free of the supernatant. This procedure was repeated four times or more until the supernatant gave a 280 nm reading below an optical density of 0.1 absorbance. The pellet of washed cells were then suspended in 3 volumes of isotonic phosphate buffer to produce 2 x 10 cells/ml. One millicurie of I was added and 20 nmol of lactoperoxidase for each milliliter of cell suspension. Ten microliter portions of 1 mM peroxide was added at 1 min intervals. The lymphocytes were then spun down at 500 g for 5 min and washed three times with phosphate-buffered saline. 

Control experiments should be included in order to determine whether the cells under investigation contain endogenous systems capable of catalyzing iodination or iodide oxidation. These controls should include a sample in which the enzyme lactoperoxidase is omitted and one to which peroxide is not added. 

Antigens, haptens, and antibodies radiolabeled with or are commonly used as tracers in immunoassay. These nuclides can be introduced directly into functional groups normally present in proteins and other macromolecules or into suitable derivatives that can be synthesized by a variety of chemical procedures. The most widely used iodination methods have been direct chemical or enzymic substitution of hydrogen in tyrosine or related groups using chloramine-T or lactoperoxidase, respectively. These methods are described in separate chapters in this volume. 

In the other method, I-labeled aniline was prepared by the chlora-mine-T method, diazotized, and allowed to react with the protein under basic conditions (Fig. 1). Presumably it couples to the phenol moiety of tyrosine residues. If this is the case, the product is analogous to that obtained by direct iodination by the chloramine-T or lactoperoxidase procedures. Papain and certain plant lectins in which cysteine residues are part of the active site were labeled without loss of activity. When variations of the chloramine-T or lactoperoxidase methods were used, 64-83% of the activity was lost. 

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