Molecular and functional identification and purification of complement component factor D from urine of patients with chronic renal failure.

Urine proteins of normal subject and patients with impaired renal function were analyzed by two-dimensional polyacrylamide gel electrophoresis. As a result, a clear spot was detected specifically in urine from patients with obvious renal dysfunction. The isoelectrical point of this unique spot was pH 7.1-7.2 and the flow-rate (Rf) was 0.50-0.55 as that of albumin was 1.0. Partial amino acid sequence analysis revealed that the NH2-terminal to 22nd amino acid sequence was identical with that of complement factor D. We purified 22 mg of this protein (factor D) from 5000 ml of urine from a patient on hemodialysis by three chromatographic steps using DEAE-Sephadex A-50 and Sephacryl S-200. The purified urine factor D gave a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis at the position of 23 kD, and displayed normal factor D hemolytic activity. The concentrations of factor D estimated by hemolytic assay were 1.9 micrograms/ml of normal serum, less than 0.1 microgram/ml of normal urine, 15 micrograms/ml of patient serum and 50 micrograms/ml of patient urine.

Isolation of complement protein D from urine of patients with Fanconi's syndrome.

Complement protein D is the least abundant of all complement proteins and, thus, one of the most difficult to purify. We report a new method for obtaining pure D from urine of patients with Fanconi's syndrome. The method is simple and allows the purification of milligram amounts of D within a few days. It involves three chromatographic steps using Bio-Rex 70, hydroxylapatite HPLC, and reverse-phase HPLC. Protein D purified by this method is suitable for both functional and structural studies.