Human a , - Microglobulin PURIFICATION PROCEDURE

A human cu,-glycoprotein of low molecular weight has been isolated from urine of patients with tubular proteinuria and shown to be present in serum and urine of healthy individuals. The isolation procedure includes ultrafiltration, gel chromatography, zone electrophoresis, and ion exchange chromatography. The yield of purified protein was 10 to 15% of the amount in concentrates of urinary proteins. The isolated protein appeared pure on immunodiffusion analyses and homogeneous when subjected to ultracentrifugation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Charge heterogeneity was observed by other electrophoretic techniques. At pH 8.6 the main part of the protein migrated in the slow a,-globulin region. cu,-Microglobulin has a brown color due to a tightly linked and unidentified chromophore material. The protein apparently consists of a single polypeptide chain that has some tendency to form noncovalently linked dimers. The molecular weight as determined by sedimentation equilibrium ultracentrifugation was 26,700, by sedimentation-diffusion data 26,100, and by gel chromatography in 6 M guanidine hydrochloride 24,800. Carbohydrate analyses revealed close to equimolar amounts of glucosamine, mannose, galactose, and sialic acid with a total carbohydrate content of 20%. The protein was’ also heterogeneous in charge after removal of sialic acid residues by digestion with neuraminidase. This suggests that the heterogeneity resides in the polypeptide chain or in the chromophore material. A relatively high frictional ratio (1.45) indicated that the molecule has an elongated shape, or a high solvation, or both. Circular dichroism spectra suggested the presence of P-pleated sheet conformation and the absence of (Y helical structure. Immunochemical determinations of cy,-microglobulin gave the following mean values: normal serum, 54 mg/liter, serum of umbilical cord blood, 26 mg/liter, and normal urine, 9 mg/24-h urine volume. Both serum and urine from patients with renal diseases had greatly increased concentrations of ru,-microglobulin. This finding and the small