Determination of molecular weight, isoelectric point, and glycoprotein moiety for the principal skin test-reactive component of histoplasmin.
A purified component designated HPD (histoplasmin-purified derivative) dII was isolated from two different cru-e histoplasmin lots by a combination of gel filtration and polyacrylamide disc electrophoresis (Sprouse, 1969). A 0.05-mug portion of HPD dII was reactive and specific in detection of delayed hypersensitivity in guinea pigs experimentally infected with Histoplasma capsulatum. The objective of this study was to characterize this skin test-reactive component for (i) homogeneity, (ii) molecular weight, (iii) isoelectric point, and (iv) composition. Sephadex chromatography, polyacrylamide disc electrophoresis, sucrose density gradient ultracentrifugation, acid and heat denaturation, and immunoelectrophoresis indicate that HPD dII is (i) homogeneous, (ii) of approximately 12,000 molecular weight, and (iii) a glycopeptide of approximately 60% carbohydrate and 40% proteinaceous composition. The marked acid and heat stability exhibited by the compound probably is attributable to the prominent carbohydrate moiety in the molecule. Isoelectric focusing indicated an isoelectric point of 5.68. This would suggest that dII is an acidic compound with either predominance of acidic amino acid residues in the molecule or, more probably, an abundance of electron donors in the carbohydrate moiety. In summary, HPD dII appears to be a glycopeptide of approximately 12,000 molecular weight, reactive in elicitation of delayed hypersensitivity of histoplasmosis.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D006168:Guinea Pigs; D006659:Histoplasmin / Q000032:analysis*; D007122:Immunoelectrophoresis; D007526:Isoelectric Point; D008970:Molecular Weight; D011489:Protein Denaturation; D012882:Skin Tests
D006023:Glycoproteins; D006659:Histoplasmin