| dc.description.abstract | The work described in this thesis can be broadly classified into two major sections namely, (1) Chemical studies, which describes the preparation of lipovitellins, fractionation of their protein components and comparison of their chemical properties (Chapter 2 and 3) and (2) Physical studies which describes the application of physical techniques like NMR, fluorescence, circular dichroism and infrared spectroscopy to study the structure of lipovitellins and VLDL (Chapter 4 and 5).
Chapter-2 describes the preparation of ?- and ?-lipovitellins by a slight modification of Wallace's procedure by TEAE cellulose chromatography. Only ?- and ?-lipovitellins were eluted from the column when a linear gradient was used. Phosvitin was not eluted under these conditions. Pure lipovitellins were obtained by rechromatography on TEAE cellulose column. Purity of the lipovitellins was tested by the determination of protein phosphorus content and SDS-PAGE of apovitellins.
The freeze-dried and salt-free lipovitellins were treated with 10% SDS containing 10?² M mercaptoethanol and their protein components were fractionated by Sephadex G-200 gel filtration. ?-lipovitellin was fractionated into ?? and ?? protein fractions, while ?-lipovitellin was fractionated into ?? and ?? fractions. ?? and ?? fractions were obtained homogeneous. ?-lipovitellin gave 7 protein bands on SDS-polyacrylamide gel. They were designated as ??, ??, ??, ??, ??, ??, ??. ?-lipovitellin gave 8 protein bands. They were designated as ??, ??, ??, ??, ??, ??, ??, ??. ?? and ?? were major protein bands of ?-lipovitellin, while ??, ?? and ?? were the major protein bands of ?-lipovitellin. ?? and ?? proteins of Sephadex G-200 correspond to ?? and ?? protein bands of SDS-polyacrylamide gel respectively.
SDS-gel pattern and Sephadex G-200 elution pattern of LDLE apoprotein was very similar to that of egg yolk VLDL apoprotein.
M.W. of ?? and ?? fractions was shown to be 30,000 and 31,000 respectively by SDS-PAGE. M.W. of ? bands range from 103,000 to 44,800, whereas ? bands range from 80,000 to 56,000. Amino acid composition, N-terminal amino acid, neutral sugars, hexosamines and protein phosphorus content of ??, ??, ?? and ?? protein fractions have been compared. Lysine was the N-terminal amino acid in all the four fractions. Neutral sugars, hexosamine and protein phosphorus content of ?? and ?? fractions were relatively more when compared with ?? and ?? fractions. Sialic acid was present only in ??. Hexosamine and protein phosphorus content was somewhat more in ?? fraction than in ??.
Protein components of ?- and ?-lipovitellins were stained on SDS-gels using phosphoprotein, glycoprotein and sialoprotein stains. Glycoprotein staining revealed that all the protein bands of ?- and ?-lipovitellins contained carbohydrates. Phosphoprotein staining of ?-lipovitellin revealed that ?? protein band is more intensely phosphorylated. Sialoprotein stain of ?-lipovitellin gave a faint blue band in the region of ?? protein. Amino acid composition of ?? and ?? fractions were similar while ?? and ?? fractions appeared to differ especially in their isoleucine content.
Physical studies:
Chapter-4 deals with the studies on pH-dependent reversible association-dissociation phenomenon of lipovitellins. The lipovitellin dimers exist at neutral pH and it dissociates when the pH is increased to the alkaline range. Lipovitellins at two different pH values, one at predominantly monomeric form and another at predominantly dimeric form was studied by proton NMR, IR, tryptophan and ANS fluorescence and by pyrene excimer fluorescence. By NMR and pyrene excimer fluorescence an increase in the mobility of choline groups and fluidity of lipids was observed when the lipovitellins were in the monomer form. IR studies indicated no major change in the secondary structure of protein. This is in agreement with the earlier observation of Franzen. In ?-lipovitellin, a splitting of resonance was observed. In the monomer form, the resonance at high field was relatively sharp and the intensity was high when compared to that in the dimeric form. Increasing the temperature of ?-lipovitellin had a large effect on the chemical shift of (-CH?-)? proton resonance. With increasing temperature the intensity of (-CH?-)? proton resonance at the high field increased with a corresponding decrease in the low field resonance. This suggests differences in the average environments of (-CH?-)? protons. On removal of 95% protein by proteolysis, ?-lipovitellin showed no splitting in the signal but gave only one proton resonance at the low field indicating that lipid-protein interaction is essential for the existence of two types of environment for (-CH?-)? protons.
The structural change associated with chloroform absorption by lipovitellins and VLDL have been studied by NMR, CD, quenching of tryptophan fluorescence, ANS binding and pyrene excimer fluorescence. By NMR and pyrene excimer fluorescence studies an increase in the mobility of choline groups and fluidity of lipids was observed during chloroform absorption. In ?-lipovitellin, the (-CH?-)? proton resonance at high field slightly increased in its intensity during chloroform absorption.
The way similar to monomerisation or at higher temperatures. CD studies indicated no major change in the secondary structure of the proteins of lipovitellins during chloroform absorption.
The interaction of Ca²? and Na? ions with VLDL as monitored by ANS fluorescence indicated Ca²? ion can enhance ANS fluorescence considerably more than Na?. Effect of pH on ANS fluorescence indicated a decrease in ANS fluorescence with an increase in the pH. These two observations are in keeping with data obtained for phospholipids. This indicates that phospholipids of VLDL are the probable binding sites of ANS.
Proteins situated on the surface of lipovitellins and VLDL were determined on polyacrylamide gel using fluorescamine as a surface label. Some preliminary data on the spatial relationship of proteins in ?-lipovitellin has been obtained using 1,5-difluoro, 2,4-dinitrobenzene as a bifunctional reagent.
ApoC of hen's egg yolk VLDL has been recombined with egg yolk lecithin or a mixture of egg yolk lecithin and triolein. Lipid-bound ApoC was separated and its lipid and protein content was determined. The lipid-bound ApoC had increased amount of helical content as compared to VLDL. | |
