Semi-Finals CHEM Past Paper PDF

Experiment 7: Isolation and Purification of Proteins The isolation of proteins from the chemical mass or biological fluid in which they are found requires normally the liberation of the cell contents by disruption of the tissue in a suitable medium. The gentlest possible method for breaking open th...

Experiment 7: Isolation and Purification of Proteins The isolation of proteins from the chemical mass or biological fluid in which they are found requires normally the liberation of the cell contents by disruption of the tissue in a suitable medium. The gentlest possible method for breaking open the cell is generally desirable. Water, dilute salt, acid, or alkaline solutions may be used to extract the proteins. Association of proteins with other organic substances may prevent the extraction of the desired protein with aqueous solvents. This requires preliminary treatment of a ground tissue suspension with weak detergent solutions or organic solvents such as acetone, ether, or butanol. RIGHT – CARBOXYLIC GROUP LEFT - AMINO WHOLE MILK COMPOSITION Vitamins Thiamine Riboflavin Panthothenic Acid Vitamin A Vitamin B12 Vitamin D Minerals Calcium Sodium Potassium Zinc Iodine Selenium Proteins Histidine - Caseins  Lysine - Lactalbumins Methionine - Lactoglobulins Phenylalanine Threonine Tryptophan Isoleucine Leucine Valine Carbohydrates Mostly Lactose Lipids Fats Protein Tertiary Structure Types Fibrous Structural proteins that are made up of long and narrow strands They ARE something Globular More compact and rounded in shape They DO something Casein - Found in milk - Binding agent - Part of a group called phosphoproteins, collections of proteins bound to something containing phosphoric acid - Also called milk solids - It can be produced skim milk by adding an acid to cause the protein to coagulate, where it can be filtered to separate the curds from the whey - Sodium caseinate is produced by reacting the acid casein with sodium hydroxide - The casein content of milk represents about 80% of milk proteins - The principal casein fractions are o Alpha (s1) o Alpha (s2)-caseins o ß-casein o kappa-casein - - Casein Composition 20.2% Glutamic Acid 6.5% Valine 5.5% Isoleucine 2.8% Histidine 1.1% Tryptophan 10.2% Proline 6.4% Aspartic Acid 4.5% Phenylalanine 2.7% Alanine 0.3% Cystine  8.3% Leucine 5.7% Serine 4.4% Threonine 2.5% Methionine 7.4% Lysine 5.7% Tyrosine 3.7% Arginine 2.4% Glycine - Structure of caseins are similar to denatured globular proteins - High # of proline residues in caseins causes particular bending of the protein chain & inhibits the formation of closed-packed, ordered secondary structures. - Contains no disulfide bonds (define) bc of the small percentage of cystine which accounts for the disulfide action in the side chains - The lack of tertiary structure accounts for the stability of caseins against heat denaturation bc there is very little structure to unfold - Without a tertiary structure there is considerable exposure to hydrophobic residues = strong association reactions of the caseins and renders them insoluble in water - Uses:  o Nutrient value o Good emulsifiers – helping fats to stay suspended in water-based products such as milkshakes, coffee creamers, and ice creams o Binder – lunchmeats, sausages, etc. o Clarifying wine – causing fine particles to coagulate with the protein so they can be easily filtered out or precipitate o Food colors – makes a nice opaque white color, which can then be tinter with other colors as required o Plastics – thin plastic films of casein can be made by adding glycerol or sorbitol as a plasticizer, a substance that lowers the temperature at which a plastic softens, and makes it more pliable. - Zwitterions: o o Mongrel ion or hybrid ion o If pH < pI = cation o If pH < pI = anion o Isoelectric point – the pH where the zwitterion is formed, protein is less soluble so more ppt o o Isoelectric Isolation ▪ pH of Calcium Caseinate = 4.6 *insoluble in solutions with a pH less than 4.6 ▪ The pH of milk is 6.6: casein has a negative charge at this pH and is solubilized as a salt ▪ If an acid is added to milk, the negative charges on the outer surface of the casein micelles are neutralized (protonation of the phosphate groups) turns to 4.4 o The casein micelles are destabilized/aggregated bc the electric charge is decreased to that of the isoelectric point (pH at which there is no net charge because there are equal numbers of positive and negative chargers present  o The casein micelles disintegrate and the casein (the neutral protein) precipitates because it is no longer polar, with the calcium ions remaining in the solution. - Isolation of Protein: o Milk is heated to 40*C (optimal temp for denaturation) ▪ Formation of curds o Acetic Acid (HCl) is added dropwise ▪ To adjust the Ph to the isoelectric point of casein ▪ This will cause the casein to “clot” and precipitate out along with butter fat leaving a liquid component called whey ▪ The liquid will change from milky to almost clear when no more casein separates. ▪ Heating the milk causes the micelles to dissociate more readily when the Ph is lowered, freeing up the amino acids. It is important to not heat the milk too hot because over the optimal temperature the curds dissociate quickly into fine particles and are no longer curds. ▪ If too much acetic acid is added The protein ppt. will dissolve ▪ The casein and butterfat are separated from the whey by straining the ppt. thru the cheesecloth ▪ Casein is insoluble in ethanol Used to remove the unwanted fat from the preparation ▪ The casein is dried by using vacuum filtration. The curds will need to be broken up by mashing to remove as much liquid as possible. ▪ Since the casein is with water. Acetone is added. Acetone is used to remove water molecules from the casein by evaporating them. Apparatus: - Hot plate - Stirring rod - Beaker - Pippete - Cheese cloth Chemical Symbols: - 10% HCl - 1% AgNO3 - Dilute HNO3  Isolation of Casein from Milk 25mL Whole Milk: evaporated milk + H2O (1:1)/skim milk + H2O (1:3) + heat (40*C) slowly over a 10 min. period + 10% HCl dropwise (w/ stirring) until a flocculent ppt. forms pH must decrease to about 4.8 Centrifuge and decant the whey FILTRATE (whey) RESIDUE Wash w/ 15mL H2O Stir vigorously and decant + 2 drops of dilute HNO3 (to prevent losing casein thru peptization) FILTRATE RESIDUE Transfer to beaker + 10mL H2O + 2 drops of dilute HNO3 FILTRATE RESIDUE Test with 1% AgNO3 (no white ppt = chloride free, white ppt = chloride present) Filter and dry in cheese cloth + 95% alc to cover Stir vigorously for 3 mins. Filter FILTRATE RESIDUE Wash with acetone Filter FILTRATE RESIDUE Weigh the dry crude extract and calculate yield in g/mL  Isolation, Hydrolysis and Qualitative Analysis of RNA from Yeast A. Isolation of Yeast RNA 5mL 1% NaOH + 25mL H2O + 5.0 dry yeast + heat for 15 min. w/ occasional stirring Strain thru cheesecloth/gauze Collect the supernatant liquid + glacial acetic acid until faintly acidic Supernate is turbid Supernate is over 10mL Centrifuge and decant Evaporate over water bath until volume is 10mL Repeat until clear liquid is obtained Cool, centrifuge and decant if necessary Cool to 40*C or lower and set aside + 0.2mL of conc. HCl to a 10mL 95% ethanol + 10mL of acidified C2H6O into the cooled mixture with vigorous stirring Divide the mixture into 2 portions in 2 separate T.T. Centrifuge and decant the supernatant liquid Wash the residue 2x w/ 2mL of 95% ethanol Centrifuge and decant after each washing Wash the residue 2x w/ of ether Centrifuge and decant after each washing result  B. Acid Hydrolysis of RNA + 10mL of 10% H2SO4 to 1 portion of the isolated RNA from yeast and cover with marble Place in a boiling water bath for 1hr + H2O to maintain the original volume Benedict’s Test Orcinol Test Test for Purine Bases Test for Inorganic Phosphate 5 drops Hydrolysate Unhydrolyzed RNA 2 drops of 2 drops of 5 drops of hydrolysate and 5 drops 5 drops of hydrolysate and 5 drops Solution hydrolysate solution unhydrolyzed RNA + unhydrolyzed RNA (separate T.T.) + 6M unhydrolyzed RNA (separate T.T.) + excess 1 drop conc. HCl NH4OH until alkaline NH4OH Neutralize w/ solid + 4 drops of Orcinol + 4 drops of Orcinol + 5% AgNO3 until ppt. occurs Acidify w/ 6M HNO3 Na2CO3 reagent reagent + 4 drops of + 4 drops of + heat in a boiling water bath for 3 min. If no ppt, let the solution stand undisturbed + 5 drops Ammonium Molybdate reagent Benedict’s reagent to Benedict’s reagent to for 5 min. 2 drops of the sol. the sol. + heat in a boiling Greenish-Blue Solution Observe under microscope Warm in a water bath for 10 minutes and water bath allow to stand Blue Solution – Blue solution result result Yellowish Green Solution Discussion: Nucleic Acids - Macrobiopolymers – high molecular weight w/ mononucleotide as the repeating unit - Information molecules - The 2 structural kinds of nucleic acids are DNA and RNA. - They are made up of nitrogenous bases, sugar, and phosphate group. - The Nitrogenous Bases - Tissues with o High nuclear = high DNA concentration o high cytoplasmic volume = high RNA value - Major components of all cells (5-15% of their dry weight) - Three types of interactions responsible for the rigid molecular configuration of nucleic acids o The phosphodiester bonds that join the nucleotides in each chain o The hydrogen bonds that join the bases o The van der Waal’s forces between stacked bases - Isolation should be conducted such that drastic changes in their structures are avoided or minimized - General procedures for isolation of nucleic acids o Disruption of the cell membrane and membrane of subcellular particles to release the nucleic acids o Treatment with a solution which will cause dissociation of the nucleoprotein and denaturation of proteins o Purification of nucleic acids - Saccharomyces cerevisiae (yeast) o Uni-cellular fungus o Contains 4% RNA by weight - Isolation of yeast RNA o The isolation of RNA from yeast involves heating with alkali (NaOH) which extracts nucleic acids and water-soluble proteins and inactives nucleases which degrade RNA o The nucleic acid us then separated from associated protein and other interfering substances by acid extraction at Ph 4-5. o Treatment with alcohol and conc. HCl can be precipitate the RNA and repeated washings with alcohol and ether/organic solvents can remove lipids. - Hydrolysis of Nucleic Acids - Ribose prefers alkali hydrolysis, - Pyrimidine are more resistant to acid hydrolysis - Purine-N-glycosyl bond is susceptible to acid hydrolysis - Hydrolysis of nucleic acid by either chemical or enzymatic methods, yields: o Purine bases (adenine & guanine) o Pyrimidine bases (cytosine, thymine, uracil) o Oligonucleotide containing up to 20 residues o Nucleosides (base + sugar) o Ribose or D-ribose o Phosphates Qualitative Tests Samples Result Analysis Hydrolysate Unhydrolyzed RNA  BENEDICT’S TEST Brick- red PPT. Blue SOL. - Aldehydes and ketones are oxidized by alkaline copper - Test for reducing sugars (+) (-) sol. The cupric ions are reduced to cuprous and brick- - red Cu2O forms. ORCINOL TEST Blue green SOL. Blue green SOL. - It is the decomposition of pentoses when heated with - Test for pentoses (+) (+) concentrated HCl to form furfural which then condenses with orcinol (3,5-dihydrotoluene) to form a blue colored compounds. - TEST FOR PURINE BASES Whitish PPT. Grayish-white PPT. - Hydrolysate + NH4 + AgNO3 = whitish - Test for guanine or adenine (+) (+) precipitate/flocculent ppt. - Unhydrolyzed + NH4 + AgNO3 = grayish-white ppt. TEST FOR INORGANIC PHOSPHATE Clear SOL. w/ yellow PPT. Yellow SOL. w/ yellow PPT. (+) (+) Objectives: 1. To perform the process of isolating RNA from yeast and conducting acid hydrolysis to the sample. 2. To conduct qualitative tests on both the hydrolyzed and unhydrolyzed RNA and analyze the results. Apparatus:  - Beakers - Stirring rod - Dropper/pipette - Cheese cloth - Graduated cylinder - Test tubes - Litmus paper - Hot Plate - Centrifuge Chemical Symbols: - 1% NaOH - H2O - C19H14O2 - CH3COOH - HCl - C2H5OH - (C2H5)2O - H2SO4 - AgNO3 - HNO3 - (NH4)2MoO4 - Na2CO3 - C7H10CuNa2O15S - C 7H 8O 2 - NH4OH Analysis: Guide Questions: 1. How can intact DNA be obtained from a solution of DNA and RNA? Obtaining intact DNA from a solution of DNA and RNA relies on the differential solubility of DNA and RNA in ethanol. DNA is less soluble in ethanol than RNA, so when ethanol is added, DNA precipitates out of solution while RNA remains in the supernatant. By carefully isolation the DNA after centrifugation, one can obtain a relatively pure DNA. How can you distinguish purines from pyrimidines via hydrolysis procedures? 2. How can you distinguish purines from pyrimidines via hydrolysis procedures? 3. Compare the results of the qualitative tests for the hydrolyzed and unhydrolyzed samples. A. Benedict's Test: Unhydrolyzed RNA: Benedict's test is used to detect reducing sugars. Unhydrolyzed RNA may show a positive result in Benedict's test due to the presence of ribose sugars in RNA molecules. Hydrolyzed RNA: After hydrolysis, the ribose sugars are broken down into their constituent monosaccharides (such as ribose). These monosaccharides are likely to give a stronger positive reaction in Benedict's test compared to the unhydrolyzed RNA, indicating the release of sugars upon hydrolysis. B. Orcinol Test: Unhydrolyzed RNA: Orcinol test is specific for detecting pentoses, which are sugars found in RNA (ribose). Unhydrolyzed RNA may show a positive result in orcinol test due to the presence of ribose sugars. Hydrolyzed RNA: After hydrolysis, the ribose sugars are indeed released as monosaccharides. The hydrolyzed RNA sample would show a stronger positive reaction in the orcinol test compared to the unhydrolyzed RNA, confirming the presence of ribose. C. Test for Purine Bases: Unhydrolyzed RNA: RNA contains purine bases (adenine and guanine) in its structure. The test for purine bases can be qualitative (colorimetric) or quantitative. Unhydrolyzed RNA would contain intact purine bases, which can be detected qualitatively. Hydrolyzed RNA: Hydrolysis breaks RNA into nucleotides and bases. The hydrolyzed RNA sample might still show a positive result in the test for purine bases, indicating the presence of adenine and guanine released from the RNA structure. D. Test for Inorganic Phosphates: Unhydrolyzed RNA: RNA contains phosphate groups in its backbone. A test for inorganic phosphates would likely show a positive result due to the phosphate groups present in RNA molecules. Hydrolyzed RNA: Hydrolysis breaks down RNA into nucleotides, releasing phosphate ions. The hydrolyzed RNA sample would also show a positive result in the test for inorganic phosphates, indicating the release of phosphate ions upon hydrolysis. Comparison Summary: Benedict's Test and Orcinol Test: Both tests would likely show stronger positive reactions in the hydrolyzed RNA sample compared to the unhydrolyzed RNA sample, due to the release of sugars (ribose) upon hydrolysis. Test for Purine Bases: Both hydrolyzed and unhydrolyzed RNA samples are expected to show positive results, indicating the presence of adenine and guanine in the RNA structure. Test for Inorganic Phosphates: Both hydrolyzed and unhydrolyzed RNA samples are expected to show positive results, indicating the presence of phosphate groups in RNA. In conclusion, hydrolysis of RNA breaks it down into its constituent components (ribose, purine bases, and phosphate groups). Qualitative tests performed on hydrolyzed RNA typically show stronger positive reactions compared to tests performed on unhydrolyzed RNA, due to the release and increased availability of these components post-hydrolysis.