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Transcript of Biochemistry: Proteins
a condensation reaction that yields a red/wine-colored solution in the presence of an oxidizing agent such as Cl2 /Br2 Techniques in Isolating and Purifying Proteins Experiment 2
brought to you by:
Garcia, Rozelle B.
Tondo, Daniel Oliver O.
Torres, Katryna Mae Ann T. Experiment!!! Conclusions,
Recommendations & Guide Questions: Introduction Protein Isolation and Purification?! How??? The experiment aims to isolate the protein albumin from an egg white and to purify the isolated proteins through qualitative tests. A Tissue contains lots of different proteins! Thank you Scientists!
I just need to isolate the protein from the other proteins and purify it from the tissue! I can't simply get the protein I like. :( But I can do it! There are various ways on how to extract a certain protein from a tissue sample. One technique is used in our
experiment. But let me tell you about
the others too! Fractional Centrifugation Column Chromatography In isolating proteins from a tissue... isolate the organelle or remove contaminating cells
extracellular proteins can be separated by salting out
for proteins inside the organelles, first the cell must be ruptured and the results homogenate that can be separated into its component organelles by fractional centrifugation. Then, the organelles may be subjected to detergent action and or sonification and may be further fractionated. Techniques of Isolating Proteins from Cells Salting Out the precipitation of proteins by raising the salt concentration separation by sedimentation of particles differing in density, shape or size at different rates in a field of centrifugal force. Based on size; the larger particles are eluted first Size Exclusion/Gel-Filtration Chromatography has a stationary and mobile phases; based on polarity Affinity Chromatography uses specific ligands; works like a lock and key Ion-Exchange Chromatography separates based on ionic charge Polyacrylamide Gel Electrophoresis (PAGE) Can separate based on charge, size, and shape same as PAGE but uses a detergent (sodiumdodecyl sulphate) that unwinds proteins and covers them with negative charges so that size is the only factor that separates them SDS-PAGE Ovalbumin Albumin Isolation of Albumin a water soluble protein
forms a colloid when combined with water kind of albumin found in egg whites
main protein in egg whites
it is a storage protein The technique employed in the experiment was Salting Out The buffer prevents the denaturation of the protein. Salting out was done by the addition of ammonium sulfate Hydrolysis is the reaction of an organic compound with water. Egg white was diluted with a 0.1M Phosphate Buffer with a pH of 7.0. Certain amounts of ammonium sulfate was added to reach a saturation of 25%, 46%, and 100%. After each addition, the resulting solution was centrifugated at 5000 rpm for 10 minutes. Isolates + HCl
(in tubes with marbles placed in hot water bath for 30 minutes and then neutralized) = Salting Out Salting In employs the differences in solubility
the salt interacts with water more efficiently than the protein thereby lessening the protein’s solubility in water, making it precipitate out of the solution
the percentage of saturation dictates how much of the protein will precipitate V.S. at low salt concentration, the solubility of the protein increases slightly
ions from the salt associate with the protein's surface, shielding those areas from the water molecules
less water molecules are required to interact with the protein and the concentration of "free" water is increased
the net effect is that the protein becomes more soluble. Amount of Ammonium Sulfate needed was calculated using an Ammonium Sulfate Calculator % Saturation Amount of
Ammonium Sulphate Precipitate
Composition 7.31 g
18.25 g 25%
100% mucoprotein globulin
and albumin albumin Qualitative Tests Often catalyzed (acidic/alkaline) Peptide bonds between amino acid molecules are results of condensation reactions. As HCl vaporizes, it comes into contact with the protein molecules, and breaks the peptide bonds. Hydrolysis Results to a mixture of the amino acids that made up the protein (hydrolates) - although in the form of their positive ions because of the presence of the hydrogen ions from the hydrochloric acid Hydrolysis Isolates and Hydrolates were dissolved in salt solution Biuret Test Only the isolates were tested using this. -detects peptide bonds
-detects the presence of proteins
-positive result is violet color of the solution test tube + 0.1 mL of distilled water (negative) / albumin (positive) /
isolates (experimental) + 1mL NaOH and 1-2 drops CuSO4 Negative: Colorless Solution
Positive: Purple solution
Isolates: Purple Solution Of course! Since the isolates contain proteins, specifically albumin! --based on the ability of Cu (II) ions to form a violet-coloured chelate complex in alkaline conditions
--For this to happen, the amino group must not be fully protonated, thereby leaving the lone pair of N to freely interact with other substances, such as the electrophilic Cu 2+ ion. This conformation is ensured only if the solution is alkaline. Biuret Test Lone electron pairs from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ion. The complex is responsible for the purple color of the solution. Xanthoproteic Test Isolates and Hydrolates were subjected to this test - test for the presence of ACTIVATED aromatic amino acids like tryptophan and tyrosine
- phenylalanine is aromatic but is not activated
- positive results is the yellow color upon addition of HNO3 and heating and when NaOH is added, the color turns orange-red. Test tubes +
0.1 mL isolates (experimental) / hydrolates (experimental) / 1% tryptophan (positive) / distilled water (negative) +
1 mL HNO3 + heat + NaOH Negative: Yellow -> no color change
Positive: Yellow -> more saturated yellow color
Isolates: Yellow -> more saturated yellow color
Hydrolates: Yellow -> more saturated yellow color The isolates contain either tryptophan or tyrosine! Nitrated Tyrosine (a) and Tryptophan (b) Nitric acid gives a color when heated with proteins containing tyrosine (yellow color) or tryptophan (orange color)
the color is due to nitration (electrophilic aromatic substitution)
the derivative formed
show an intensely yellow
color Xanthoproteic Test Phenylalanine is not activated because it has a benzene ring which is very much stable. Sakaguchi Reaction Isolates and Hydrolates were tested using this. - detects arginine
- positive result is a reddish wine color of the solution Test tube +
0.1 mL isolates (experimental) /
hydrolates (experimental) /
1% arginine (positive) /
distilled water (negative) +
2mL NaOH + 2mL Molisch reagent. + 0.5 mL Bromine water Negative: dark yellow green color
Positive: wine red color
Isolates: wine red color
Hydrolates: wine red color Albumin contains arginine residues! Sakaguchi Reaction Proteins can be isolated using the method of salting out. After a protein has been isolated, it is subjected to hydrolysis and then to different qualitative tests to detect the presence of certain amino acids.
In the experiment, the first isolate contained mucoproteins while the second and third contained ovalbumin and globulin.
Arginine and tryptophan are the amino acids present in ovalbumin. hydrolysis because it is an essential step for identifying amino acids. Other techniques which can be used in isolating and identifying proteins and/or amino acids are chromatography and gel electrophoresis. These techniques can be used to yield more accurate results.
In this particular experiment, some of the results did not coincide with the theoretical results (particularly in the qualitative analysis). We recommend future performers of the experiment to use a more efficient way of hydrolysis because it is an essential step for identifying amino acids. Why must the sample be tested with the Biuret test be sufficiently alkaline? What other techniques can be used to separate proteins of varying molecular weights, charge and polarity? Explain briefly. What structural feature must be present in an amino acid or substance to get a positive result for the xanthoproteic test? Explain the principle involved in isolating proteins by fractional precipitation. What is salting out? Why is buffered (NH4)2SO4 used instead of just a solution of the salt? Garrett, R.; Grisham, C. (2010). Biochemistry, 4th ed. Boston:Brooks/Cole, Cengage 2010.
Laboratory Manual in Biochemistry. (n.d.). Comittee on Biochemistry, Department of
Physical Sciences and Mathematics, College of Arts and Sciences, University of
the Philippines Manila.
Biuret Test. (n.d.) [online source] Retrieved from: www.biosci.ohiou.edu/introbioslab/Bio170/170_2/biuret.htm.
Uniprot: Ovalbumin. (n.d.) [online source] Retrieved from: www.uniprot.org/uniprot/
Reference.com: Sakaguchi Test Amino Acid Proteins. (n.d.) [online source] Retrieved
from: www.reference.com/motif/science/sakaguchi-test-amino-acid-proteins. References: Fractional precipitation works using the concept that different proteins have different solubilities and will precipitate at different concentrations.
Salting out involves the addition of a hydrophilic salt to a mixture which contains protein to reduce the solubility of that protein in the mixture.
The addition of the buffer prevents drastic changes in the pH and reduces the chance of the protein denaturation. Explain the principle involved in isolating proteins by fractional precipitation. What is salting out? Why is buffered (NH4)2SO4 used instead of just a solution of the salt? A positive reaction for this test is due to the presence of an activated aromatic group in the protein molecules. What structural feature must be present in an amino acid or substance to get a positive result for the xanthoproteic test? Other techniques that could be used are gel electrophoresis, and chromatography.
Chromatography separates substances into the stationary and mobile phases. There are three types. One is Partition Chromatography, used for separating proteins based on differences in the phase distribution of the substances between insoluble phases. Another is Adsorption Chromatography, which separates mixtures by selective adsorption onto the surface of a solid material, and Gel Filtration Chromatography, which can separate substances based on differences in molecular weight. What other techniques can be used to separate proteins of varying molecular weights, charge and polarity? Explain briefly. Gel electrophoresis is a method for separating and analyzing proteins based on their size and charge. It uses a gel with charges electrodes on opposite ends. Positively-charged proteins tend to travel to the negative end more and the negative proteins to the positive end. And since the substance is placed on gel, heavier molecules tend to move slower toward the poles than the smaller ones. What other techniques can be used to separate proteins of varying molecular weights, charge and polarity? Explain briefly. For the violet complex to appear, the amino group must not be fully protonated, thereby leaving the lone pair of N to freely interact with other substances, such as the electrophilic Cu 2+ ion. This conformation is ensured only if the solution is alkaline. Why must the sample be tested with the Biuret test be sufficiently alkaline? TABLE 1. Amount of Solid Ammonium Sulfate Added to
Buffer Solutions of Different Saturations TABLE 2. Experimental Positive and Negative Control Group Results for
Qualitative Tests Used for Isolates and Hydrolysates Identification TABLE 4. Composition of the Resulting Precipitates
from Each Round of Centrifugation TABLE 3. Experimental Results for the Qualitative Tests Used for
Isolates and Hydrolysates Identification Experimental Results Only the results for the Sakaguchi reaction did not confer with the theoretical results. This may be due to inadequate time for hydrolysis.