ovalbumin and 2-iminobiotin

Topics: Avidin; Biotin; Chromatography, Affinity; Indicators and Reagents; Ovalbumin; Sepharose

Topics: Acetylgalactosamine; Avidin; Biotin; Cell Membrane; Galactose; Galactose Oxidase; Indicators and Reagents; Membrane Proteins; Ovalbumin; Sialic Acids

A general method for the selective retrieval of surface labeled plasma membrane components had been devised. The basis of the technique is the covalent attachment of compounds containing 2-iminobiotin, the cyclic guanidino analog of biotin, onto the cell surface proteins and the use of immobilized avidin to recover the labeled components uncontaminated by other cytosolic and membrane components. The pH-dependent interaction of 2-iminobiotin with avidin makes recovery possible. At high pH the free base form of 2-iminobiotin retains the high affinity specific binding to avidin characteristic of biotin, whereas at acidic pH values, the salt form of the analog interacts poorly with avidin. Model studies on the interaction of 2-iminobiotinylated proteins with avidin-Sepharose 4B show that for tight binding to the affinity matrix, the pH of the column must be 9.5 or higher, that a single 2-iminobiotin group is sufficient for binding, and that proteins with different extents of labeling behave similarly when the low pH buffer is applied. When intact human erythrocytes were sequentially labeled with periodate and 2-iminobiotin hydrazide and the Triton X-100-solubilized plasma membrane proteins were subjected to affinity isolation, the major sialoglycoproteins, periodic acid-Schiff (PAS) 1, PAS 2, and PAS 3, plus two proteins with apparent molecular weights higher than band 3 were retrieved. The recovery of these proteins is not due to a nonspecific adsorption to the affinity matrix.

Topics: alpha-Fetoproteins; Avidin; Biotin; Cell Membrane; Erythrocyte Membrane; Glycoproteins; Humans; Hydrogen-Ion Concentration; Membrane Proteins; Methods; Ovalbumin; Protein Binding; Spectrophotometry

Topics: Avidin; Biotin; Carbon Radioisotopes; Chromatography, Affinity; Ovalbumin; Rhodamines; Sepharose; Tritium