Summary
By coupling purified horseradish peroxidase with glutaraldehyde and subsequent reaction with insulin, conjugates were obtained. These were partially purified by ion exchange chromatography on DEAE-cellulose. A fraction with an average molar ratio of peroxidase to insulin 1:0.37 was analysed by electrophoresis and gel filtration. The peroxidase activity of this fraction was found to be 19% of normal and the immunological reactivity 0.6% as compared with that of insulin.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Avrameas, S., Uriel, J.: Methods de marquage d'anticorps avec des enzymes et son application en immunodiffusion. C.R. Acad. Sci. (Paris)262, 2543 (1966).
Avrameas, S.: Coupling of enzymes to proteins with glutaraldehyde. Immunochemistry6, 43–52 (1969).
Avrameas, S., Ternick, T.: Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry8, 1175–1179 (1971).
Brill, A.S., Weinryb, I.: Reactions of horseradish peroxidase with azide. Evidence for a methionine residue al the active site. Biochemistry6, 3528–3535 (1967).
Chance, B., Maehly, A.C.: In: Colowick, S.P. and Kaplan, N.O. Methods in enzymologie2, p. 769. New York: Acad. Press 1955.
Cuatrecasas, P.: Insulin-Sepharose immunoreactivity and use in the purification of antibody. Biochem. biophys. Res. Commun.35, 531–537 (1969).
Ellis, W.D., Dunford, B.: The effect of ligand binding and acid splitting on the rotatory dispersion of ferric horseradish peroxidase. Canad. J. Biochem.46, 1231–1235 (1968).
Korn, A.H., Feairheller, S.H., Filachione, E.M.: Glutaraldehyde: Nature of the reagent. J. Mol. Biol.65, 515–529 (1972).
Maehly, A.C.: Splitting of horseradish peroxidase into prosthetic group and protein as a means of studying the linkages between hemin and protein. Biochim. biophys. Acta8, 1–17 (1952).
Meade, R.C., Klitgard, H.M.: A simplified method for immunoassay of human serum insulin. J. Nucl.Med.3, 407–416 (1962).
Nakane, K.P., Pierce, G.B.: Enzyme labeled antibodies: Preparation and application for localization of antigens. J. Histochem. Cytochem.14, 929 (1966).
Nakane, P.K., Pierce, G.B.: Enzymes labeled antibodies for the light and electron microscopic localization of tissue antigens. J. cell. Biol.33, 307–318 (1967).
Richards, F.M., Knowles, J.R.: J. Mol. Biol.37, 231–233 (1968).
Shannon, L.M., Kay, E., Lew, J.Y.: Peroxidase izoenzymes from horseradish roots. J. biol. Chem.241, 2166–2172 (1966).
Shnitka, T.M., Seligman, A.M.: Some recent electron chemical application of diaminobenzidine (DAB) technic. Ann. rev. Biochem.40, 375 see 390–396 (1971).
Spackman, D.H., Stein, W.H., Moore, S.: Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chemistry30, 1190–1206 (1958).
Author information
Authors and Affiliations
Additional information
Kindly supported by Landesversicherungsanstalt Rheinprovinz, Düsseldorf, and Landesamt für Forschung, Nordrhein Westfalen.
Rights and permissions
About this article
Cite this article
Biener, J., Jansen, F.K. & Brandenburg, D. Insulin labelled by coupling with peroxidase. Diabetologia 9, 53–55 (1973). https://doi.org/10.1007/BF01226002
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01226002