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A single column cation exchange method was developed which allowed simultaneous recovery of lysozyme and avidin from undiluted egg white using a unique elution sequence which involved accumulation of avidin on the column through several cycles of egg white application and lysozyme elution. Lysozyme was recovered with higher yields than reported for the isoelectric precipitation methods often used in the industry (86% vs 60 - 80%) and in high purity. Avidin recovery was also as good or better than that of previously reported ion exchange methods (74 - 80% vs 17 -80%). The purity of the avidin fraction (up to 40.9%) was superior to other reported primary avidin fractions. Avidin was shown to have a greater potential for both electrostatic and hydrophobic interactions with Duolite C-464 than lysozyme but under the conditions of this separation, electrostatic interactions were dominant. Secondary purification of avidin by carboxymethyl celluose cation exchange (CMC), gel filtration, metal chelate interaction chromatography (MCIC), aliphatic hydrophobic interaction chromatography (HIC), and Phenyl-Sepharose interaction chromatography (PSIC) each resulted in considerable increase in avidin purity. In terms of resin capacity, yields, and avidin purity however, CMC ion exchange was superior. A comparison of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andnative protein electrophoresis profiles gave clear evidence of protein - protein interaction between avidin and lysozyme in partially purified avidin preparations. This interaction may also occur between the native proteins in the egg white, but has not been demonstrated with certainty. The molar ratio of avidin to available biotin binding sites was estimated by 5 methods. For highly purified avidin samples the hydroxy azo benzoic acid (HABA) method proposed by Green (1965) was superior. A new method, utilizing an immobilized biotin column, which did not require extensive purification of avidin was found to give similar results. Finally, a highly sensitive assay of proteins bound to nitrocellulose membranes was developed which was capable of quantifying as little as 0.12 µg of protein. Membrane bound proteins were labeled with peroxidase via a biotin - avidin linkage as previously reported and bound peroxidase activity was related to initial protein content. The method was applicable to determination of the relative concentrations of different protein bands on Western blots of electrophoresis gels. / Land and Food Systems, Faculty of / Graduate

A lysozyme from Bacteriophage 7v was purified 7.7 fold over the original lysates of the bacteriophage 7v and Pseudomonas aeruginosa PS-7. This purification process includes ultracentrifugation, ammonium sulfate precipitation, dialysis, and fractionation in a Sephadex G-150 column. The phage lysozyme exhibits a greater specificity when assayed with P. aeruginosa cells as a substrate, but still is capable of acting on the standard lysozyme Micrococcus lysodeikticus substrate. The pH optimum, heat inactivation range, and action on other bacteria is described. The molecular weight was found to be 14,300. The values of this 7v phage lysozyme are in close agreement with values found with other phage lysozymes. A possible treatment for burn wounds infected with Pseudomonas aeruginosa is also described.

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Call number: LD2668 .T4 1963 M88 / Master of Science

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Graduation date: 1999

Graduation date: 1997