Investigation of immunoadsorbent efficiency and capacity for the isolation of tRNAs containing N6-(delta 2-isopentenyl)adenosine derivatives.
Antibodies directed to modified nucleosides recognize the nucleoside (antigen) when it is present in an intact tRNA molecule. The general application of anti-nucleoside immunoadsorbent chromatography, however, has been greatly impeded by the apparent inefficiency and low capacity of conventional immunoadsorbents for transfer RNA. Antibodies specific for isopentenyladenosine (i6A) were employed to investigate the efficacy of various immunoadsorbents with respect to immobilization of antibody protein and with respect to their ability to bind i6A-containing tRNAs. Biologically active anti-i6A was recovered in high yield (80-88%) by affinity chromatography on i6A-adipate-Sepharose 4B or i6A-butane diglycidyl ether-Sepharose 4B using either 15% pyridine in phosphate-buffered saline or 0.2 M acetic acid as eluents. The binding capacity of various anti-i6A antibody immunoadsorbents was evaluated. While both anti-i6A antibody-protein A-agarose-iminothiolane (ITL) and anti-i6A antibody-protein A-agarose-dimethyl suberimidate showed a high capacity for i6A-tRNA, the latter column is much less efficient with respect to antibody immobilization. Under optimal conditions, the ITL immunoadsorbent binds 5-6 nmol of i6A/mg of antibody protein. With respect to bulk tRNA, 1 mg of antibody protein (ITL immunoadsorbent) binds all of the i6A-tRNA in a 1-mg sample.
D000241:Adenosine / Q000031:analogs & derivatives*; D000936:Antigen-Antibody Complex; D001419:Bacteria / Q000032:analysis; D007106:Immune Sera; D005779:Immunodiffusion; D007163:Immunosorbent Techniques; D007164:Immunosorbents; D007541:Isopentenyladenosine / Q000032:analysis* / Q000276:immunology; D012343:RNA, Transfer / Q000302:isolation & purification*; D011863:Radioimmunoassay
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D007164:Immunosorbents; D007541:Isopentenyladenosine; D012343:RNA, Transfer; D000241:Adenosine