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Author Pinnamaraju, Susmitha, author.
Title Synthesis and bee-venom PLA2 catatlyzed hydrolysis of a fluorescently labeled phosphatidylcholine analogue / by Susmitha Pinnamaraju.
Published [Northridge, California] : California State University, Northridge, 2011.
LOCATION CALL # STATUS
 Electronic Book  QD40 .Z952 2011 P56eb    ONLINE
  
Description 1 online resource (x, 60 pages) : charts, graphs, black and white.
Content Type still image
text
Format online resource
File Characteristics text file PDF
Thesis M.S. California State University, Northridge 2011.
Bibliography Includes bibliographical references (pages 55-60).
Summary Fluorescently labeled mixed chain phosphatidylcholine was synthesized and used for kinetic studies of bee-venom phospholipase A2. Preparation of the phospholipid compound with chain-terminal fluorescent groups with a donor-acceptor pair has been accomplished. L-α,β-isopropylidene-glycerol was used to provide the chiral glycerol skeleton, tetrahydropyranyl and phenoxyacetyl protecting groups were employed to achieve orthogonal protection at sn-2 and sn-3-glycerol positions respectively. The phosphocholine function was introduced using 2-chloro-2-oxo-1,3,2-dioxaphospholane with anhydrous trimethylamine. The sequence included formation of lysophosphatidylcholine intermediate which was acylated to introduce the sn-2-acyl group with the chain terminal second fluorophore. The rates of bee-venom PLA2 acting on the fluorescent phosphatidylcholine analogue incorporated in mixed micelles with dodecyldimethylammonio propane sulfonate (DPS) have been determined using standard pH-stat method at pH 8.00, 38°C, in the presence of 10 mM CaCl2. Under these experimental conditions complete hydrolysis of the synthetic phospholipid analogue was achieved. Enzymatic hydrolysis of on (2 mM: 8mM) PC (30)-DPS mixed micelles resulted in activity of 257 µmol/ min/ mg and (2mM: 8mM) DPPC-DPS mixed micelles occurred with 679 µmol/ min/ mg. The enzymatic activities for a series of assay mixtures containing mixed lipid system, [PC (30), DPPC] at fixed lipid-to-surfactant ratio (2mM: 8mM) were also measured. The kinetic data indicate that the structurally modified phosphatidylcholine (30) was hydrolyzed by bee-venom phospholipase A2 with the specific activity about 3 times lower than the natural substrate (DPPC). The activity vs. mole fraction of PC (30) solubilized in DPS mixed micelles plot indicated that the rate of hydrolysis decreases linearly with increase in PC (30) portion in (2mM: 8mM) mixed phosphatidylcholine-DPS mixed micelles. This linear decrease of the activity in mixed lipid system was compared with the calculated activity as a linear variation between the values of 279 at XPC (30) = 1 and 679 at XPC (30) = 0. The linear decrease in activity of bee-venom PLA2 on the mixed lipid system exhibits close to ideal behavior which shows that even though PC (30) is structurally modified at the chain terminals with spectroscopic labels this, has only minor effect on the substrate properties of the mixed micelles when natural substrate DPPC is replaced with PC (30). The spectroscopic properties of the synthetic phospholipid analogues have been determined prior to the kinetic studies of hydrolysis of the functionalized compound by bee-venom phospholipase A2 enzyme.
Note Description based on online resource; title from PDF title page (viewed on December 09, 2011).
Local Subject Dissertations, Academic -- CSUN -- Chemistry and Biochemistry -- Biochemistry.
OCLC number 849950483