Comparative studies on radiolabeling of lysozyme by iodination and reductive methylation.
Before attempting to radiolabel proteins it is essential that conditions be found for optimal reaction by use of cold reagents. Iodination by the chloramine-T method was not suitable for radiolabeling of lysozyme as it resulted in reduced solubility, large conformational changes, loss of enzymic activity and a decrease in immunochemical reactivity. On the other hand, reductive methylation of lysozyme by formaldehyde and sodium cyanoborohydride was considered suitable for radiolabeling of lysozyme. The extent of reaction with the free amino groups was dependent on the concentration of lysozyme and the molar ratios of the reactants (lysozyme, NaCNBH3 and HCHO). The molecular weight, net charge and enzymic activity of the lysozyme derivatives were similar to the native molecule. The immunochemical reactivity was reduced by 6-13% when more than 6 amino groups were modified. Reductively methylated rabbit IgG showed unaltered molecular weight, net charge and very little conformational changes compared to native IgG. Partial reaction, by reductive methylation using [14C]HCHO, lysozyme with specific activity of 11.1 X 10(6) cpm/mg protein and pig anti lysozyme antibody with specific activity of 2.95 X 10(5) cpm/mg protein were prepared.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001894:Borohydrides; D002700:Chloramines; D005557:Formaldehyde; D007074:Immunoglobulin G / Q000378:metabolism; D007455:Iodine / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D008745:Methylation; D009113:Muramidase / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013552:Swine; D014105:Tosyl Compounds
D000906:Antibodies; D001894:Borohydrides; D002700:Chloramines; D007074:Immunoglobulin G; D014105:Tosyl Compounds; C016300:chloramine-T; D005557:Formaldehyde; D007455:Iodine; C009282:sodium cyanoborohydride; D009113:Muramidase