Detection of lysozyme and alpha 2-macroglobulin—lysozyme complexes by immunoblotting

Abstract

An immunoblotting technique was developed to detect human lysozyme and lysozyme complexes in body fluids. The unoccupied binding capacity of proteins was demonstrated by addition of surplus lysozyme. The sensitivity of immunoblotting to the free enzyme' in human albumin solution was < 5 ng. In serum and pleural fluid, part of exogenous lysozyme was bound to alpha 2-macroglobulin (α 2-M). At high concentrations of lysozyme in leukemic sera, part of the enzyme formed an endogenous α 2-M complex. On the other hand, the formation of α 2-M complexes with exogenous lysozyme was especially striking in sera from nephrotic patients with elevated α 2-M. The findings corroborate with previous reports on lysozyme binding to purified α 2-M in vitro and suggest that the binding is concentration-dependent with respect to both reaction partners. In vivo the mechanism may provide a pathway for extrarenal lysozyme catabolism mediated by reticuloendothelial cells. No other binding proteins were seen in the present study: lysozyme did not bind to serum immunoglobulins in 35 samples with an immunoglobulin paraprotein, three samples with polyclonally elevated gamma-globulins, 20 other patient sera and 10 normal sera. Neither did lysozyme bind to urinary proteins in five samples from patients with myeloic leukemias nor in 10 samples from myeloma patients with urinary excretion of a monoclonal immunoglobulin light chain.