A Method for Determining the Sedimentation Behavior of Enzymes: Application to Protein Mixtures

Markedly increased quantities of lysozyme have been found in the serum and urine (ranging to 2.6 g per day) of ten consecutive cases of monocytic and monomyelocytic leukemia. The enzyme has been isolated from the urine of several cases and physicochemically and immunochemically characterized. It is apparently identical to the lysozyme of normal tears, saliva, leukocytes, and serum, but structurally different from the lysozyme of hen's egg white. The activity of the human enzyme assayed with M. lysodeikticus organisms is 3 to 12 times greater than egg white lysozyme at equivalent concentrations. An agar plate method has been developed for quantitating lysozyme activity in small samples (approximately 25 µl) of serum, urine, or other biological fluids. The range and reproducibility of this method were found to be superior to previously available lysozyme assay procedures. Present evidence indicates that lysozyme is the principal, if not the sole, product of the proliferating monocytes in monocytic and monomyelocytic leukemia, and quantitation of serum and urine lysozyme should be a useful diagnostic procedure for these leukemias.

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Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia

Some asparagine and glutamine residues in proteins undergo deamidation to aspartate and glutamate with rates that depend upon the sequence and higher-order structure of the protein. Functional groups within the protein can catalyze this reaction, acting as general acids, bases, or stabilizers of the transition state. Information from specific proteins that deamidate and analysis of protein sequence and structure data bases suggest that asparagine and glutamine lability has been a selective pressure in the evolution of protein sequence and folding. Asparagine and glutamine deamidation can affect protein structure and function in natural and engineered mutant sequences, and may play a role in the regulation of protein folding, protein breakdown, and aging.

Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins.

Publisher Summary This chapter focuses on vertebrate lysozymes, particularly hen egg-white (HEW) lysozyme. Lysozymes of other types generally are distinguishable from HEW lysozyme in having higher molecular weights and somewhat different enzymic activities. Lysozyme is small and basic, and it separates well on weak acid resins, like Amberlite XE-64 or Bio-Rex 70 or carboxymethyl cellulose and on calcium phosphate gel. Affinity chromatography of lysozyme has been carried out using dispersed chitin or carboxymethyl (CM)-chitin. The conditions required for elution from chitin columns suggest that there are two classes of adsorbent sites that are differently affected by pH and ionic strength in their interaction with the enzyme. The preparation of lysozyme from diverse animal sources is generally achieved in four main steps, which include (1) the preparation of a lysozyme-rich extract, (2) chromatography on CM-cellulose, (3) filtration on Sephadex G-25, and (4) ion exchange chromatography on Amberlite CG-50 at 20° C with a 0.2 M phosphate buffer. The chromatography is sensitive to the pH and small variation of no more than 0.1 pH unit can involve complete retention or exclusion.

21 Vertebrate Lysozymes

Publisher Summary This chapter discusses the deamidation of glutaminyl and asparaginyl residues in peptides and proteins. The occurrence of glutaminyl and asparaginyl residues in peptides and proteins is so widespread that these residues have become known as 2 of the 20 most commonly occurring amino acid residues. It has been hypothesized that these residues play static roles in protein structure and also play dynamic roles as molecular timers of biological events. Deamidation of glutaminyl and asparaginyl residues has been responsible for artifacts in peptide and protein chemistry. The rate of deamidation of these residues has been shown to be dependent upon primary sequence, secondary and tertiary structure, temperature, pH, ionic strength, and special intermolecular interactions. Deamidation has been found to occur in a wide variety of different proteins and may be expected to occur in most proteins under physiological conditions. Many of the essential processes in living things may be timed by deamidation of glutaminyl and asparaginyl residues. These molecular timers are under simple genetic control, are adjustable to many different timed intervals, and are easily implementable by means of the important changes that they can effect in the proteins in which they are included.

L.R. Wetter

Papers

Immunological studies on egg white proteins. IV. Immunochemical and physical studies of lysozyme.