A Radioimmunoassay for the N-terminal Propeptide of Rat Procollagen Type III: Application to the Study of the Uptake of the N-terminal Propeptide of Procollagen Type III in Isolated Perfused Rat Liver

Abstract

Antibodies against a synthetic peptide representing the 14 C-terminal amino acids of the N-terminal propeptide of rat and bovine procollagen type III were raised in rabbits and used to develop a radioimmunoassay. N-Terminal propeptide of procollagen type III, purified from calf skin, served as standard and tracer material. The IC50 of the standard inhibition curve was 2.1 μ/I, the lower limit of detection about 0.4 μg/1, interassay variation was 8.5% and the intraassay variation 6.6% in typical experiments. Three peaks of antigenicity were detected in rat serum after gel chromatography. One peak coeluted with purified N-terminal propeptide of procollagen type III, one peak contained material approximately twice this size, and one peak eluted close to the void volume of the column. The antigen concentration in rat serum decreased in an age dependent manner. Rat, bovine, sheep and minipig serum antigen was sufficiently crossreactive to allow the application of the assay to these species, whereas human, goat and guinea pig samples were not. The degradation product Col 1, causing non-parallel inhibition in commercially available assays for human samples was not recognized since it does not contain the epitope represented by the synthetic peptide. The assay was used to study the half-life of bovine and endogenous N-terminal propeptide of procollagen type III in isolated perfused rat liver. [125I]-labeled antigen was cleared rapidly from the perfusate (t1/2 less than 5 min). The bovine antigen was removed from the perfusate with a half-life of 15 ± 4 min. Endogenous propeptide was perfused for 120 min with little change in concentration. The data indicate that the studies of elimination of peptides by the liver may be influenced by species differences, and that iodinated proteins are not a suitable tool for such studies.