Six of the ten cases had evidence of renal and electrolyte abnormalities, particularly hypokalemia and hyperkaluria. The mechanisms of these abnormalities and their possible relationship to the excretion of CP are under investigation, and detailed clinical, hematologic, and cytologic studies will be published subsequently.Blood and urine samples from 20 normal subjects and 250 patients with a wide variety of disease states, including other forms of leukemia, infectious mononucleosis, multiple myeloma and other plasma cell dyscrasias, renal diseases, tuberculosis and other chronic infections, sarcoidosis, carcinoma, and Hodgkin's disease were also studied. Fresh serum, separated from the clot within 2 hr of collection or EDTA (Versene), plasma samples were used. Heparinized plasma is unsatisfactory because of the formation of complexes between heparin and lysozyme.Normal tears were obtained from D. O., age 10, provoked by her two older siblings.Protein Quantilation.--24-hr urinary collections were obtained and no preservative was added. Aliquots of whole urine were concentrated 40-to 50-fold by dialysis against 25% polyvinylpyrollidone (PVP). Total urinary protein excretion was determined by precipitation with 5% trichlor~cetic acid and Kjeldahl analysis. Standard procedures were employed for the electrophoretic (cellulose acetate, Spinco) analyses and for the starch gel and acrylamide gel electrophoretic studies. Ultracentrifugal analyses were performed with the Spinco Model E ultracentrifuge, and sedimentation constants calculated to infinite dilution ($20, w).Isolation of CP.--The urinary proteins, including CP, were initially precipitated from 24-hr collections by 60% saturation with ammonium sulfate. The precipitates were suspended in water and dialyzed against phosphate-buffered saline, pH 7.2, until free of ammonium ion. Saline insoluble material was discarded and the saline solution of protein dialyzed against 0.1 ~ NaOH:glycine buffer, pH 9.5. Chromatographic separation of CP from other urinary proteins was achieved by DEAE (diethylaminoethylcellulose) with the 0.1 ~¢ Na glycinate buffer, by using either column or batch separation techniques. Under these conditions, CP was recovered in the void volume, and the other urinary proteins were retained by the DEAE.More recently we have used the procedure of Alderton, Ward, and Fevold (2) for the isolation of lysozyme using bentonite adsorption and elution with 5% aqueous pyridine adjusted to pH 5.0 with sulfuric acid.Peptide Analyses.--Tryptic peptide maps were made with the cooperation of Dr. FrankTischendorf by the procedure of Katz, Dreyer, and Anfinsen (3) with minor modifications. DEAE-isolated CP samples were oxidized with performic acid and precipitated with 10% trichloroacetic acid. The precipitates were washed with trichloroacetic acid, absolute ethanol, and ether. The oxidized CP samples were incubated for 4 hr with trypsin (Worthington Biochemical Corporation, Freehold, New Jersey, 2 X crystallized trypsin SF, 1 mg per 100 mg protein) in 0.2 ~ NH4HC...
