Masters Theses

Date of Award

8-1986

Degree Type

Thesis

Degree Name

Master of Science

Major

Physics

Major Professor

Solon Georghiou

Abstract

Nanosecond emission anisotropy using 1,6-diphenyl-1,3,5- hexatriene (DPH) as the fluorescent probe, the enhancement of the 0-0 vibronic transition in the fluorescence spectrum of pyrene (which serves as a measure of the microenvironment of the probe), and a membrane fusion assay employing chlorophyll a and chlorophyll b as indicators of the mixing of membrane contents, have been used to investigate the effects of bee venom melittin on dimyristoylphosphatidylcholine (DMPC) unilamellar and multilamellar liposomes.

Nanosecond emission anisotropy studies performed on DMPC unilamellar vesicles at 16°C, 33°C and at the transition temperature, in the absence of melittin and in the presence of melittin at a protein to lipid molar ratio of ~1/60 have shown that at all three temperatures melittin brings about an increase in the order of the membrane's acyl chains and a reduction in the rate of diffusion of the probe molecule. This effect is most noticeable below the transition temperature. The same measurements made on multilamellar vesicles have shown that the protein causes a decrease in order only at the transition temperature, and does not affect the rate of diffusion at any of the temperatures investigated.

Measurements of the enhancement of the forbidden 0-0 vibrational transition in pyrene were performed for pyrene in unilamellar and multilamellar vesicles, in the absence of the.protein and in its presence at a relative concentration of ~1/60, over the temperature range from approximately 5°C to 35°C. For sonicated samples it was found that melittin enhanced the 0-0 vibronic transition of pyrene at 16°C, 33°C and at the lipid transition temperature. This indicates that the protein brought about an increase in the order of the mem brane and a decrease in its fluidity which allowed the probe molecule to migrate closer to the glycerol backbone of the lipid molecules. This interpretation is consistent with the nanosecond emission anisotropy data. It was also found that melittin induced a sharpening of the gel-to-liquid crystalline transition, implying that the protein increased the cooperativity of the lipid molecules. Similar experiments performed on multilamellar vesicles under the same conditions showed that melittin caused little change in the enhancement of the 0-0 vibrational transition at 16°C and 33°C. In the vicinity of the transition temperature, however, melittin was found to greatly decrease the strength of the enhancement, implying that the membrane's order had decreased, which allowed the probe to migrate further towards the nonpolar center of the bilayer. Results of the nanosecond emission anisotropy and the 0-0 vibrational enhancement measurements were again found to be consistent. The measurements using pyrene also indicated that melittin broadens the gel-to-liquid crystalline transition in multilamellar vesicles, thus reducing the cooperativity of the lipid molecules.

Finally, a membrane fusion assay, which measures energy transfer efficiency between chlorophyll a and chlorophyll b incorporated into the membrane, was used to determine that, at the protein-to-1ipid molar ratio used, melittin caused the fusion of approximately three unilamellar liposomes when the latter were incubated with melittin at 33°C. No such fusion was found to occur in the case of the multilamellar vesicles.

The results of these studies show that melittin exerts its greatest effect on the unilamellar vesicles, where it alters the order and fluidity of the membranes, and presumably their morphology as well. The changes in the order should be viewed as the collective effect of a number of different melittin-induced processes which have taken place in the unilamellar vesicles.

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