Photocleavable biotinylated ligands for affinity chromatography.
Two derivatives of the peptide hormone cholecystokinin (CCK-8s) containing a photocleavable o-nitrobenzylester group have been synthesized. One analog, [3H]4-(biotin-epsilon-Ahx-oxymethyl)-3-nitrobenzoyl-Gly-Orn (propionyl)-epsilon-Ahx-CCK-8s ([3H]BANA-CCK-8s), was biotinylated, while the other, 4-alanyloxymethyl-3-nitrobenzoyl-epsilon-Ahx-CCK-8s (ANA-CCK-8s) had a free amino group for coupling to amino-reactive affinity matrices. The analogs retained high affinity to both CCK receptors and anti-CCK antibodies. [3H]BANA-CCK-8s was bound to a streptavidin-agarose affinity matrix and was subsequently released by irradiation with uv light of wavelengths > 320 nm. The ability to recover a biotinylated peptide ligand from a streptavidin column under very mild conditions should be useful in the purification of specifically binding proteins. As a demonstration of this approach, [3H]BANA-CCK-8s was incubated with anti-CCK-antiserum and was subsequently passed over a streptavidin-agarose affinity matrix. After washing, the bound antibodies were eluted by photocleavage of the affinity ligand. Forty-six percent of the bound antibodies were recovered after 40 min of irradiation. The eluted antibodies showed essentially unchanged binding characteristics. The suitability of ANA-CCK-8s for the purification of anti-CCK antibodies was also demonstrated. The approach may prove to be generally useful in the isolation of labile proteins in an intact form.
Thiele
C
C
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Fahrenholz
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin / Q000378:metabolism*; D002766:Cholecystokinin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D008024:Ligands; D008027:Light; D008969:Molecular Sequence Data; D010777:Photochemistry / Q000379:methods*; D011817:Rabbits; D011949:Receptors, Cholecystokinin / Q000378:metabolism; D012685:Sepharose; D019809:Streptavidin
D001426:Bacterial Proteins; D008024:Ligands; D011949:Receptors, Cholecystokinin; C089131:4-(biotin-epsilon-(aminohexanoyl)oxymethyl)-3-nitrobenzoyl-glycyl-(propionyl)ornithinyl-epsilon-aminohexanoyl-cholecystokinin; C089130:4-alanyloxymethyl-3-nitrobenzoyl-epsilon-aminohexanoyl-cholecystokinin; D001710:Biotin; D002766:Cholecystokinin; D012685:Sepharose; D019809:Streptavidin