Isolation of Human Urinary Lysozyme

Masuda, Nobuhito; Kobayashi, Shizuko; Mizuno, Katsuya; Sakiyama, Fumio

doi:10.1093/oxfordjournals.jbchem.a132211

Cited by 2 publications

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“…Although all of the initial enzyme activity was recovered in the purified material, the possibility still exists that the non-adsorbed fraction contains antigenically masked and/or enzymatically-inactive denatured lysozyme which would not be detected in our assay system. Both the parotid and leukemic enzyme were found to have an amino-terminal lysine residue in agreement with reported studies for the leukemic enzyme [25,26] and the amino acid compositions were very similar to those previously determined by other investigators [1,2,25,26]. The homogeneity of the parotid enzyme was further confirmed by sedimentation equilibrium ultracentrifugation.…”

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confidence: 90%

Quantitative Recovery, Selective Removal and One‐Step Purification of Human Parotid and Leukemic Lysozymes by Immunoadsorption

Mackay

Iacono

Zuckerman

et al. 1982

European Journal of Biochemistry

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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-activited Sepharose 6B. Lyophilized parotid saliva (2 1) 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.The use of classical biochemical techniques for the isolation of lysozyme from either human parotid or whole saliva have to date resulted in the purification of small quantities of homogeneous enzyme preparations [ 1-31. Attempts to improve the yields of purified enzyme through one-step systems based on affinity chromatography methodology have, by and large, been unsuccessful [4,5]. Recently, Vasstrand and Jensen [6] published a two-step procedure employing Bio-Rex 70 and chitin affinity chromatography which resulted in good recovery of apparently highly purified and homogeneous lysozyme from human whole saliva. Although their technique is simple and relatively rapid, the necessity of employing Bio-Rex 70 to obtain high yields of enzyme negated its potential value for antibacterial studies in which lysozyme would be selectively removed from saliva and then reconstituted with the entire lysozyme-depleted salivary fraction. Bio-Rex 70 is known to bind avidly a group of cationic polypeptides which are difficult to elute and recover in an active form and which may be important to the antibacterial function of saliva [7a].As an alternative and one-step approach to permit both purification with high recoveries of enzyme and selective removal of lysozyme for future salivary antibacterial studies simultaneously, immunoadsorption affinity chromatography has been investigated. An immunoadsorbent of sheep anti-(human leukemic lysozyme) IgG was therefore prepared by coupling the IgG to epoxy-activited Sepharose 6B in order to purify lysozyme to homogeneity from parotid saliva. This Data presented in this paper have been taken in part from the dissertaion of one of us (BJM) submitted to the Graduate School, State University of New York at Stony Brook, in...

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