Biochemical Analysis: Lipids, Nucleic Acids
Isolation and Purification of Lecithin from Egg Yolk
Egg yolks are a rich and convenient source of lecithin. They contain water, fats, proteins, phospholipids, cholesterol, pigments, and vitamins. Using acetone extraction, most triacylglycerols, pigments, and vitamins can be removed, leaving mainly protein and phospholipid residue. Further extraction with chloroform-methanol solvents can remove the protein and provide a crude egg lecithin solution. Finally, lecithin can be separated from other contaminants using a silica gel column to achieve a purity of 95%.
Detecting Steroids and Their Esters
The reaction products of cholesterol with strong acids produce colored complexes with ferric salts.
Determination of Total Cholesterol
The apoB-containing lipoproteins in the specimen are reacted with a blocking reagent that renders them non-reactive with the enzymatic cholesterol reagent under the conditions of the assay.
Determination of Phosphorus in Phospholipids
Phospholipids heated with perchloric acid are degraded to CO2, H2O, and PO43-. In the acidic environment, the orthophosphate reacts with ammonium molybdate (VI) to form ammonium phosphomolybdate (VI). This compound, under the influence of reducing agents, is reduced to the mixed oxides of molybdenum, called molybdenum blue (MO2 ยด MO3), the amount of which is determined colorimetrically.
Determination of Total Lipids by Phospho-Vanillin Assay
Lipids having unsaturated bonds react with sulfuric acid to form carbonium ions, which subsequently react with the vanillin phosphate ester to yield a purple complex that is measured photometrically. Cholesterol has one unsaturated bond and is used as a standard in lipid determination. There are many methods for lipid determination, but the phospho-vanillin assay is considered the best due to its simplicity, accuracy, and low cost. This method is reliable and fast but only works for lipids containing unsaturated fatty acids. The phospho-vanillin assay is a ratio method, and the calculated lipid content may not be equal to the actual content. By extracting lipids from a sample and determining the lipid content by weight, followed by the phospho-vanillin assay, one can determine the factor to multiply to obtain the actual lipid content in the sample.
Detection of Lipid-Amino Groups
Ninhydrin reacts with free amino groups present in aminolipids and certain phospholipids.
Detection of Resorcinolic Lipids
Phenolic compounds react with diazonium salt (Fast Blue B) to give azo dyes.
Phospholipid Concentration in Solutions
Stewart Method
The Stewart method determines phospholipid concentration by exploiting the ability of phospholipids to form a complex with iron thiocyanate that is soluble in chloroform. This method can be used to determine the concentration of phospholipids in solutions containing inorganic phosphate, such as phosphate-buffered saline (PBS).
However, a limitation of this method is that the ability of individual phospholipids to form the complex can vary, so a calibration curve must be prepared using the same phospholipids and mixtures. Additionally, determining the concentration of phosphatidylglycerol is challenging because it has a low ability to form the complex with iron thiocyanate.
Electrophoresis
Electrophoresis in agarose gel and polyacrylamide gel: DNA electrophoresis in agarose and polyacrylamide uses the fact that at pH 7.0, nucleic acids are polyanions. Nucleic acids migrate from the negative electrode (cathode) to the positive electrode (anode).
DNA Separation Due to Size:
- Classical electrophoresis in agarose: from about 50,000 base pairs (bp), or 50 kbp, to about 100 bp.
- Pulsating electrophoresis in agarose: from 50 kbp to above 2-3 Mbp.
- Electrophoresis in polyacrylamide gel: from several base pairs up to approximately 1-2 kbp.
Agarose
Agarose is a polysaccharide polymer formed by galactose derivatives derived from marine algae. Its concentration ranges from 0.5% to 2%. The size of the pores depends on agarose concentration; a higher concentration results in smaller pores, making it more suitable for separating smaller molecules.
Polyacrylamide Gel
Polyacrylamide gels typically consist of acrylamide, bisacrylamide, and a buffer with an adjusted pH. A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED, are added to initiate polymerization. The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two acrylamide molecules.
Factors Influencing DNA Migration Speed
The speed of DNA migration in the gel during electrophoresis depends on:
- The size of the molecule: Linear dsDNA migrates in the gel at a rate inversely proportional to the number of base pairs (inversely proportional to the log10 of molecular weight).
- DNA conformation: The supercoiled circular (CCC), open circular (OC), and linear (LIN) forms migrate in the gel at different rates.
- Agarose concentrations
- Electric voltage
Possible DNA isolation from the gel.
Ethidium Bromide vs. Midori Green
Advantages of Midori Green:
- Midori Green binds less aggressively to DNA molecules, so fewer mutations occur during synthesis.
- Midori Green does not penetrate the skin or the plasma membrane, so uptake into living cells is minimal.
- Midori Green has a very short half-life, so it breaks down in the environment quickly.
Nucleic Acid Separation Methods
Sedimentation
- Sedimentation of nucleic acids during centrifugation (or ultracentrifugation) in density gradient solutions, e.g., cesium chloride in the presence of ethidium bromide (bacterial genomic DNA from plasmid DNA).
- Or a buffer containing various amounts of polyethylene glycol.
Chromatographic
- Various kinds of chromatographic molecular sieves.
- The longer the molecule, the faster it will “flow out” from the column.
Digestion of DNA with DNAse I
DNAse I is an endonucleolytic enzyme specific for double-stranded DNA. The high molecular weight DNA is degraded (digested) by DNAse I into smaller and smaller fragments with the duration of the reaction or the availability of the enzyme. As a result of digestion, we obtain a mixture of DNA fragments of various sizes (lengths), which in agarose gel electrophoresis may be arranged in the form of a path color even along its entire length (smear – DNA molecules “smeared” along the entire length of the path).
Determination of the Hyperchromic Effect
By heating DNA solutions in a boiling water bath, we induce thermal denaturation. Gradually cooling the denatured DNA to room temperature causes its renaturation. Rapid cooling of the DNA in an ice bath fixes the denatured form.
Determination of RNA Concentration
Orcinol Test
Ribose, under the influence of concentrated hydrochloric acid, is dehydrated into furfural, which, in the presence of Fe3+, gives a green color complex with orcinol. This reaction is provided by free ribose, its phosphate esters, and ribose of purine nucleotides and nucleosides. The pyrimidine-associated ribose does not form this complex. Therefore, when quantifying RNA, a standard RNA solution is used instead of a free ribose template.
With orcinol, in addition to ribose, deoxyribose also reacts; however, it gives about 10 times lighter color than ribose.
Dische’s Test for Deoxyribose
Deoxyribose heated in a strongly acidic environment dehydrates to a furfural that forms a blue complex with diphenylamine. Ribose and other pentoses do not give this reaction.
Bial’s Test for Ribose
Free pentoses, their phosphate esters, and ribose bound in nucleosides and purine nucleotides heated with concentrated hydrochloric acid dehydrate to furfural which, with orcinol and Fe3+ ions, gives a green-colored complex. Deoxyribose and ribose associated with pyrimidine bases give a lighter color.