Purification of human procollagen type I carboxyl-terminal propeptide cleaved as in vivo from procollagen and used to calibrate a radioimmunoassay of the propeptide.
We purified human procollagen type I carboxyl-terminal propeptide (PICP) that had been cleaved as in vivo from procollagen. PICP in serum-free medium from cultured human fetal fibroblasts was purified by thiophilic adsorption chromatography, low-pressure gel filtration, and HPLC gel filtration. The purity and homogeneity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequencing showed that the sequences of the alpha 1 and alpha 2 chains of this PICP were identical to those of the PICP produced in vivo. The monocomponent PICP thus purified was used as calibrator in a simple equilibrium-type RIA of PICP with polyclonal antibodies raised in rabbits. The measuring range is 0.15-3.75 nmol/L, and the assay detection limit is 0.03 nmol/L. The within-run and total CVs are 2% and 4%, respectively. The reference interval for the plasma concentration of PICP in healthy women of ages > 30 years is 0.36-1.44 nmol/L (geometric mean 0.72 nmol/L, n = 154).
Pedersen
B J
BJ
Center for Clinical & Basic Research, Ballerup, Denmark.
Bonde
M
M
D000328:Adult; D000595:Amino Acid Sequence; D002138:Calibration; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D005260:Female; D005347:Fibroblasts; D006801:Humans; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000097:blood; D010446:Peptide Fragments / Q000097:blood / Q000302:isolation & purification*; D017697:Premenopause / Q000097:blood; D011347:Procollagen / Q000097:blood / Q000302:isolation & purification*; D011863:Radioimmunoassay / Q000592:standards* / Q000706:statistics & numerical data; D011933:Reagent Kits, Diagnostic; D012016:Reference Values; D012680:Sensitivity and Specificity; D017421:Sequence Analysis
D010446:Peptide Fragments; D011347:Procollagen; D011933:Reagent Kits, Diagnostic; C042813:procollagen type I carboxy terminal peptide; D015675:Osteocalcin