Radioiodinated Peptides and Growth Factors and Their Applications

Abstract

The use of octadecasilyl silica and other resin columns (C₁₈-, C₈-, CN-, and phenyl resin) in the batch extraction of tissue homogenates provides a simple means of rapid purification of peptides from tissues or plasma. High acidity (5% formic acid, 1% trifluoroacetic acid, or 1 N HCl) inhibits the peptidase activity present in tissue. Immediate freezing and homogenization in cold dilute acid (pH 2) could preserve physiologically active peptides and inhibit the artificial generation of components. Polypropylene tubes, pipettes, and tips are more useful because many peptides are adsorbed to glass surface. Proteins with molecular weight greater than 40,000 are usually excluded by 6 to 10 nm pore of commercial resin. The high resolution of the separation technique is enhanced by using acidic solvent systems. At low pH, the absorption of peptides to the silica matrix is reduced, and column efficiency, in terms of peak shape and theoretical plates, is improved. In addition, hydrophobic and ion repairing agents, such as trifluoroacetic acid, are used to associate with the free amino groups, which become fully ionized at low pH. This increased affinity for the reverse phase support material permits separation of melanocyte stimulating hormone, melanocyte stimulating hormone, ACTH, calcitonin, cytochrome c, bovine serum albumin, bovine prolactin, and bovine growth hormones from brain tissue by a linear gradient of 20% to 60% acetonitrile containing 0.1% trifluoroacetic acid during a period of 2 hours. The solvent system also permits monitoring for ultraviolet light absorbance at 210 nm. Because of the volatility of the solvent, further separation and the processing of the biologically active peptide and evaluation by radioimmunoassay are possible.