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I. Purification and Partial Characterization of Testicular Hyaluronidase. II. Mechanistic Studies of Human Lysozyme


Borders, Charles LaMonte (1968) I. Purification and Partial Characterization of Testicular Hyaluronidase. II. Mechanistic Studies of Human Lysozyme. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/7RZ0-5W42.


NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. PART I A new method is presented for the purification of testicular hyaluronidase involving ion exchange chromatography followed by gel filtration on Sephadex G-75. A highly purified hyaluronidase preparation has been obtained which contains 45,000 N. F. activity units per mg dry weight and which migrates as a single component on polyacrylamide gel electrophoresis at pH 4.3. The amino acid composition has been determined, and the enzyme has been shown to be a glycoprotein containing 5.00% mannose and 2.17% glucosamine (expressed as N-acetylglucosamine). The molecular weight of the purified hyaluronidase has been determined to be 61,000 by gel filtration methods. This value is in contrast to a molecular weight of 126,000 for the crude enzyme, as determined by similar techniques. A glycopeptide has been isolated from the purified hyaluronidase after total enzymatic digestion of the reduced carboxymethylated enzyme. This glycopeptide was shown to have molecular weight of approximately 1,600, and to have a composition of (mannose)7(glucosamine)2(asp)1. The amino acid involved in the protein-carbohydrate linkage is thus aspartic acid. PART II It has been shown that when human lysozyme catalyzed glycosidic bond cleavage of chitobiose is carried out in the presence of methanol, transglycosylation occurs with production of methyl-N-acetyl-[beta]-D-glucosaminide. Thus, retention of configuration occurs at the anomeric carbon of the disaccharide substrate during catalysis and eliminates the possibility of a single displacement mechanism for the enzyme. Use has been made of this transglycosylation reaction to enzymatically synthesize oligomeric p-nitrophenyl-[beta]-D-glucosaminides from chitin oligosaccharides, p-nitrophenyl-N-acetyl-[beta]-D-glucosaminide, and human lysozyme. Such saccharides serve as substrates for human lysozyme with release of p-nitrophenol. Similar synthesis of oligosaccharides from chitin oligomers, p-nitrophenyl-[beta]-D-glucoside, and human lysozyme has been carried out. Since human lysozyme effects hydrolysis of the glucosidic bonds in such oligosaccharides, a mechanism involving anchimeric assistance of an N-acetyl group has been eliminated. Similar enzymatic synthesis and hydrolysis of oligosaccharide substrates from chitin oligomers and p-nitrophenyl-2-deoxty-[beta]-D-glucoside has eliminated a mechanism involving acetamido or C-2 oxyanion participation by substrate due to general base catalysis by the enzyme. Only two closely related possibilities remain to explain the mechanistic pathway of substrate during catalysis by human lysozyme. The hydrolysis of chitobiose, chitotriose, and chitotetraose by human lysozyme under different sets of conditions has also been investigated. It was found that this enzyme could be distinguished from hen lysozyme by the product distributions obtained for hydrolysis by the two enzymes under identical reaction conditions. The hydrolysis of p-nitrophenyl-[beta]-D-chitobioside by human lysozyme was shown to follow Michaelis-Menton kinetics, with K[subscript M] = 2.6 x 10[^-2] M and k[subscript cat] = 6.8 x 10[^-5] mole x sec[^-1] x (mole enzyme)[^-1].

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:(Chemistry)
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Raftery, Michael A.
Thesis Committee:
  • Unknown, Unknown
Defense Date:13 May 1968
Record Number:CaltechETD:etd-09262002-152327
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:3776
Deposited By: Imported from ETD-db
Deposited On:27 Sep 2002
Last Modified:18 Jul 2023 23:09

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