FURTHER PURIFICATION AND CHARACTERIZATION OF SERUM-PROTEINS USED TO DETECT CYSTIC-FIBROSIS GENOTYPES BY ISOELECTRIC-FOCUSING

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  • 1 January 1976
    • journal article
    • research article
    • Vol. 34  (1) , 51-71
Abstract
Sera from cystic fibrosis (CF) homozygotes and obligate heterozygotes contain a CF factor (.gamma. CF factor) not found by isoelectric focusing in thin-layer polyacrylamide gels in most normal control sera. Sera from most obligate heterozygotes lack another protein (band B, C, or D) that is commonly found in sera from most normal and cystic fibrosis individuals. A standardized, biophysical assay is described that employs isoelectric focusing for the detection of both CF homozygotes and heterozygotes based on the analysis of whole serum for the presence of the .gamma. CF factor and bands B, C and D. Results of analyzing sera from selected CF patients by isoelectric focusing indicated that there is a general correlation between the amount of the .gamma. CF factor and the clinical severity of the disease. Partial purification and characterization of the .gamma. CF factor and protein bands B, C and D was accomplished by using DEAE-cellulose chromatography, Sephadex G-200 gel filtration, sequential molecular filtration through a series of Amicon Diaflo ultrafiltration membranes, affinity chromatography and cellulose acetate electrophoresis. The .gamma. CF factor is a cationic protein with a pI of 8.46 .+-. 0.05, has .gamma. electrophoretic mobility, a MW between 3500 and 10,000, and apparently exists in CF serum in 2 forms (free in solution and complexed to immunoglobulinG). Bands B, C and D are cationic proteins with pI values of 7.85-8.10, have .gamma. electrophoretic mobility and a MW of approximately 100,000-150,000.