Abstract
Human complex-forming glycoprotein, heterogeneous in charge (protein HC) has previously been isolated from urine and immunochemically shown to be present in low and high molecular weight forms in blood plasma (Tejler, L., and Grubb, A. O. (1976) Biochim. Biophys. Acta 439, 82-94). In the present work, the major low and high molecular weight forms of the protein were isolated from plasma by immunosorption followed by gel chromatography. The plasma low molecular weight protein HC and the urinary protein had similar, if not identical, molecular weight, amino acid composition, NH2-terminal and carboxyl-terminal amino acid sequences and electrophoretic mobility. The low molecular weight plasma protein HC carried a yellow chromophore like the urinary protein, but its molar extinction coefficient at 280 nm was lower and its charge heterogeneity less pronounced than that of urinary protein HC. The plasma high molecular weight protein HC had a hydrodynamic volume which was greater than that of monomeric IgA but smaller than that of dimeric IgA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated high molecular weight protein followed by electrophoretic blotting and immunochemical analysis demonstrated that the protein contained four polypeptide chains: two light immunoglobulin chains (Mr = 23,000), one IgA alpha-chain (Mr = 54,000), and one chain with Mr approximately 90,000 which carried both alpha-chain and protein HC antigenic determinants. Whether the protein HC X IgA complex is a functionally significant part of the humoral immune system cannot be decided without further experimentation, but the complex was found to be completely absent from the blood plasma of patients with a selective deficiency of IgA-secreting immunocytes. The isolated low and high molecular weight plasma protein HC components were used as standard proteins in the construction of a quantitative crossed immunoelectrophoretic assay for the simultaneous quantitation of the two major protein HC components in blood plasma. The plasma concentrations of the low and high molecular weight protein HC components were measured by this method in 13 healthy Caucasians. The results for the low molecular weight protein HC were: mean, 20.3 mg/liter, S.D., 3.2 mg/liter, range, 13.6-26.0 mg/liter; and for the protein HC X IgA complex: mean, 293 mg/liter, S.D., 176 mg/liter, range, 36-620 mg/liter.
Highlights
Anders 0.Grubb, CarlosLopez$, Lars Tejlerfj, and EnriqueMendez$ From the Departmentsof Clinical Chemistry and §Medicine, Universityof Lund, Malmo General Hospital, S-214 01 Malmo, Sweden and the SSeruicio de Endocrinologia, Centra “Ramony Cajal,” Carretera de Colmenar, Km. 9.1, Madrid 34, Spain
Themajor low and highmolecular weight forms of the protein wereisolated from plasma by immuno- Human complex-formingglycoprotein,heterogeneousin sorption followed by gel chromatography.The plasma charge is a recently described LMW glycoprolow molecular weight protein HC and the urinary pro-tein originally isolated fromnormal human urine[1].It shows teinhadsimilar, if notidentical,molecularweight, a considerable charge heterogeneity, carries an unidentified amino acid composition, NH2-terminal and carboxyl- yellow-brown chromophore and hasbeen immunochemically terminal amino acid sequences and electrophomreot-ic demonstrated to occur in normal human plasma, both as a bility
Glycoproteinsa,-microglobulin and a,microglycoprotein, The plasma highmolecular weight protein HC had a both of which were isolated from the urine of patients with hydrodynamic volume which was greater than thaotf renal tubular dysfunction [2,3,4,5]
Summary
3 ml a t 4 "C by pressure ultrafiltration using an Amicon Diaflo cell (Amicon Corp., Danvers, MA). The. England), nitrocellulose membranfeilters (0.2 pm) were from immunoreactivity of the fractions for protein HC, IgA, and albumin. The analyses methyl ketone andcarboxypeptidase A and carboxypeptidase Y were were performed in aBeckman 121" analyzer equipped with a from Merck AG, BSA (Fraction V) was from Miles Laboratories Ltd. Beckman 126 data system. Half-cystine was determined eitheras (Stoke Poges, Slough, England), proteinA was from Pharmacia Fine carboxymethylcysteine or as cysteic acid after performic acid oxida-. Tryptophan was determined by hydrolysis with 3 M pdiochemical Centre (Amersham, England), Coomassie brilliant blue toluene sulfonic acid [18]. Amino Aeid Sequence Analysis-Automated amino acid sequence ylene glycol 6000 was from KEBO AB (Stockholm, Sweden), and all analysis according tothe method of Edmanand Begg [19] was reagents used in automatic sequencing were from Beckman Instru- performedusingaBeckman.
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