Growth hormone covalently bound to sepharose or glass. Analysis of ligand release rates and characterization of soluble radiolabeled products.
Purified boveine growth hormone labeled enzymatically with iodine-125 was covalently coupled to cyanogen bromide activated Sepharose 4B gel and to diazotized zirconia-clad glass beads. Under the conditions employed, an average of 0.8 and 7.3 mg of hormone were bound per ml of Sepharose and glass, respectively. When the conjugates were incubated in Krebs-Ringer bicarbonate buffer (pH 7.4), three separate radioactive species were detected in the incubation supernatant by chromatography on Sephadex G-75. The elution volumes of two of the species were identical with those of 125-I-labeled growth hormone and Na-125I controls, while the third component eluted as a moleucle of intermediate size. The rate of release of each species from the solid matrix was linear with time over 4 days and increased with temperature from 4 to 37 degrees. Although significantly less growth hormone was released from glass (0.14%/day) than from Sepharose (0.40%/day) at 37 degrees, active hormone in amounts sufficient to be detectable in a biological assay was nevertheless liberated from the former after as little as 4 hr of incubation. By contrast, the rate of release of 125-Iminus- and the intermediate-size compound from glass was significantly greater than from Sepharose, suggesting that protein bound to glass supports is more susceptible to degradation from exposure to ionizing radiation.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D003979:Diazonium Compounds; D005720:Gamma Rays; D005898:Glass; D013006:Growth Hormone / Q000031:analogs & derivatives / Q000528:radiation effects; D007457:Iodine Radioisotopes; D008024:Ligands; D008722:Methods; D011134:Polysaccharides; D011830:Radiation Effects; D012685:Sepharose; D013696:Temperature; D013997:Time Factors
D003979:Diazonium Compounds; D007457:Iodine Radioisotopes; D008024:Ligands; D011134:Polysaccharides; D013006:Growth Hormone; D012685:Sepharose; D003488:Cyanogen Bromide