Rami Almog

The use of Coomassie brilliant blue for critical micelle concentration determination of detergents

Abstract The spectral changes of Coomassie brilliant blue G-250 dye have been studied in the presence of detergent micelles. Association of detergents with the dye produced an absorbance change at 618 nm. At cetyltrimethylammonium bromide concentrations well below the critical micelle concentration a large decrease in absorbance is observed. This is due to formation of a 1:1 water insoluble complex between the anionic dye and the cationic detergent. At sufficiently low dye concentration, the 618-nm absorbance is significantly increased with micelle concentration of the detergent. The onset of enhanced absorbance may be used to determine the critical micelle concentration of cetyltrimethylammonium bromide. The critical micelle concentration of anionic, neutral, and zwitterionic detergents has been determined also by plotting the absorbance at 618 nm as a function of detergent concentration.

Stability of sonicated aqueous suspensions of phospholipids under air

The stability of phospholipids in liposomal aqueous suspension against oxidative degradation in air was investigated using spectrophotometric indices, glutathione peroxidase reactivity and thin layer chromatography. Zwitterionic phospholipid was found to be susceptible to degradation via oxidation of polyunsaturated hydrocarbon chains and ester hydrolysis, producing oxidized lysophosphatide and free fatty acid derivatives. These products were characterized as hydroperoxides based on their reactivity with the selenium-dependent glutathione peroxidase isolated from human erythrocytes. Lecithin in Tris buffer was more resistant to hydrolysis than in water. The sonication of 8.0 mM of soybean phosphatidylcholine (SB-PC) suspension in 0.1 M Tris (pH 7.5) in the presence of air produced relatively high concentration of conjugated diene hydroperoxide, but a small amount of hydrolyzed products. Anionic phospholipids, such as egg-phosphatidylglycerol (egg-PG), demonstrated higher resistance to air oxidation than the zwitterionic lecithin, but its oxidation was promoted by sonication.

The role of polar groups in the interaction of dipalmitoyl phosphatidylcholine with chlorophyll a in monolayers

Abstract Investigation of pure dipalmitoyl phosphatidylcholine (DPPC) monolayers suggests that interactions between DPPC head groups are important to the surface behavior of these molecules. Surface pressure-area studies of mixed monolayers of DPPC with chlorophyll α or β-carotene emphasize the importance of interactions between polar groups in chlorophyll α and the head group of DPPC. These interactions may play an important role in the transport of ions or electrons through membranes.

A sensitive assay for proteins and biliproteins.

Abstract An assay for proteins in dilute solution is described which is based on binding of Coomassie blue G-250 to proteins. The dye-protein complex formed is separated out from solution by centrifugation. The absorbance of the redissolved precipitate in 70:30 ( v v ) Tris:methanol is monitored at 605 nm. The assay is standardized for biliprotein determination.

Surface properties of phycocyanin

Abstract The change of surface pressure (π) during adsorption and compression of phycocyanin film at the air-water interface indicates that this phycobiliprotein is strongly attracted to the interface. It behaves like a flexible molecule but is not completely unfolded. The steep slope of the curve of π as a function of the bulk concentration ( C b ), when ( C b −3 mg/ml, may be attributed to the high number of hydrophobic amino acid residues and other structural features of the protein. At ( C b > 10 -3 mg/ml the π vs ( C b curve is interpreted as being characteristic of aggregation of the protein. Analysis of the curves of π vs ( C b and π vs molecular area ( A ) indicate that the phycocyanins tetrapyrrole chromophores make only a small contribution to the surface properties of the protein.

Interaction of C-phycocyanin with lipid monolayers under nitrogen and in the presence of air

The surface interaction of C-phycocyanin with lipids was studied using the monolayer technique. The surface activity of the protein was found to be higher at the lipid-water interface than at the nitrogen-water interface, particularly at high surface pressures of the lipid monolayer. The maximum initial surface pressures beyond which phycocyanin could not penetrate the dipalmitoylphosphatidylcholine and monogalactosyldiglycerol monolayers were 27 and 30 mN m-1, respectively. Below these values the protein demonstrated preferential interaction with the monogalactosyldiglycerol monolayer. The surface properties of the unfolded protein at pH 2.5 at the lipid-water interface were compared with those of the protein at pH 7.0. Higher affinity of the three-dimensional structure of the protein to lipid monolayers was observed, in particular by high subphase protein concentration. When the lipid films were subjected to oxidation stress by exposure to air, the surface properties of C-phycocyanin and dipalmitoylphosphatidylcholine were not greatly affected but the surface activity of monogalactosyldiacylglycerol was reduced dramatically by autoxidation. The oxidation of monogalactosyldiacylglycerol could not be prevented by the introduction of C-phycocyanin molecules at the lipid-water interface.

Effect of acetic acid on phycocyanin-phycoerythrocyanin mixture extracted from Anabaena variabilis.

Exposure of a phycocyanin-phycoerythrocyanin mixture extracted from Anabaena variabilis to sodium acetate, pH 3.8, ionic strength of 0.1, results in dissociation of the phycoerythrocyanins beta subunit from its alpha subunit. The alpha subunit obtained by this method has a strong absorption transition at 508 nm. This transition is a consequence of the subunits specific conformation, rather than of a new chromophore. The behavior of the phycocyanin-phycoerythrocyanin mixture in acetate buffers of variable compositions suggests that interactions which involve carboxylic amino acid residues play an important role, along with hydrophobic associations, in the association of phycoerythrocyanin subunits into monomers (alpha beta) and between this protein and phycocyanin. This work also indicates that the linkage between alpha and beta subunits of phycoerythrocyanin is labile and may be weaker than the association of these subunits with phycocyanin under acidic conditions.

Effects of neutral salts on the circular dichroism spectra of ribonuclease a and ribonuclease s

The circular dichroism (CD) spectra of ribonuclease A, ribonuclease S, and N-acetyltyrosineamide were recorded as a function of pH in the presence of various concentrations of inorganic salts. Above pH 9.0 salting-in of tyrosine residues increases their intramolecular associations. This association enhances the contribution from these residues to the CD spectrum leading to an apparent titration curve that is shifted toward lower pH. The data indicate that unfolding of ribonuclease A and S by inorganic salts does not begin with disrupting existing electrostatic interactions. But, as the unfolding process progresses, disruption of electrostatic interactions may take place. This is consistent with our previous calorimetric studies which suggest that unfolding of ribonuclease A by salts proceeds initially by energetically favorable solvation of the folded protein. An increase in ellipiticity at 275 nm of partially unfolded protein in salt was observed as the pH was changed from 7.0 to 4.0. This observation may suggest that the isothermal unfolding of the protein by salts at low pH proceeds through an intermediate step which involves histidine residues and causes a conformational change in the tyrosines asymmetric environment.

A study on the positive ellipticity in the circular dichroism of ribonuclease A

The small positive ellipticity near 239 nm in the CD spectrum of RNase has been investigated as a function of pH. Theoretical calculations using CD parameters representing buried or exposed tyrosine residues have been carried out. A comparison of the theoretical calculations with experimental data suggests that the changes in the bands intensity, as a function of pH, arise mainly from electronic transitions associated with the tyrosine residues. The buried tyrosine residues are the major contributors to the ellipticity in this region at neutral pH. At higher pH contributions from exposed residues are also observed.