Quantitative recovery, selective removal and one-step purification of human parotid and leukemic lysozymes by immunoadsorption.

Immunoadsorption affinity chromatography was used to isolate and purify human lysozyme. The immunoadsorbent was prepared by coupling sheep anti-(human leukemic lysozyme) IgG to epoxy-activated Sepharose 6B. Lyophilized parotid saliva (21) was resuspended in distilled water (325 ml, 50 mg/ml, w/v) and applied to a column which had a capacity to bind 4.25 mg human enzyme. Non-adsorbed material did not contain lysozyme, as determined by enzymatic and immunological analyses. All lysozyme activity present in the applied sample (1.97 mg) bound to and was desorbed from the column by elution with 0.2 M sodium acetate HCl buffer, pH 1.8. The isolated material was homogeneous as determined by cationic and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, ultracentrifugation, amino acid and amino-terminal analyses, and immunoelectrophoretic analysis. The one-step purification procedure yielded a 1370-fold increase in specific activity. Human lysozyme was also selectively purified by this method from an ammonium sulfate precipitate of the urine of a patient with chronic monocytic leukemia. Amino acid and polyacrylamide gel electrophoretic analyses indicated that the purified enzyme was identical to human lysozyme isolated from leukemic urine by classical biochemical techniques.