ACTIVITY 1A SUBCELLULAR COMPONENTS OF THE CELL

ACTIVITY 1B QUALITATIVE TEST FOR THE CHEMICAL COMPONENTS OF THE CELL

OBJECTIVES:
1) To fractionate the liver tissue and understand the idea of fractionation. 2) To identify the presence of different biochemical components of the organelles. 3) To determine the major classes of molecules.

Test for Carbohydrates

Molisch Test Benedicts Test

Molisch Test

Control: 1% Ribose solution Positive Result: PURPLE ring between acid and sediment Principle: when sugar solution mixed with alphanaphthol is brought in contact with conc. H2SO4, a violet ring is formed at the junction of the 2 liquids

Molisch Test
o Molisch test is a general test for all carbohydrates and any compound containing a carbohydrate residue in the molecule. The test is very sensitive and a dilution as low as 0.001% still gives definite positive result. o H2SO4 acts as dehydrating agent forming furfural derivatives which interact with alpha-naphthol liberating a colored compound.

Molisch Test

Molisch test shows that all types of carbohydrates gives positive result which leads to a purple color. In the experiment, the purple ring appeared to all of the sediments and the supernate but with varying degree. It is in the sediment 3 and supernate 3 that yielded the most obvious and clear purple ring while the sediment 2 yielded a faint purple ring color.

Benedicts Test

Control: 1% Ribose Positive Result: BRICK RED precipitate

Benedicts Test
The Benedict's test allows us to detect the presence of reducing sugars (sugars with a free aldehyde or ketone group). All monosaccharides are reducing sugars; they all have a free reactive carbonyl group. Some disaccharides have exposed carbonyl groups and are also reducing sugars. Other disaccharides such as sucrose are non-reducing sugars and will not react with Benedict's solution. Starches are also non-reducing sugars. The copper sulfate (CuSO4) present in Benedict's solution reacts with electrons from the aldehyde or ketone group of the reducing sugar to form cuprous oxide (Cu2O), a red-brown(brick red) precipitate.

Benedicts Test

Benedicts test shows that all monosaccharide and disaccharides reduce weak oxidizing agents that changes color from blue to reddish brown. The monosaccharide and disaccharides also give a positive result except sucrose. In our experiment, almost all of the sediments solution turned to light aqua while the control solution is reddish brown. Therefore, all of tests for the sediments and the supernate yielded negative result.

Test for Proteins

Biuret Test Xanthoproteic Test

Biuret Test

Control: 1% Albumin Positive Result: PURPLE coloration

Biuret Test
o The biuret test is a chemical test used for detecting the presence of peptide bonds. o It is a general test for proteins. A positive reaction is obtained with all native proteins and most of the derived proteins. A violet color is obtained with long chain proteins while a pink color is obtained with shorter chain. This is due to the varying amount of peptide linkage. o Alkaline solution of proteins treated with copper sulfate results in the production of a rose-pink to violet, then purple. o a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution

Biuret Test

Biurets test says that a violet color will appear in the solution if the amide bond common to proteins is present and this reaction carries out a high ph. Therefore protein with peptide bonds is present in the nucleus.

Xanthoproteic Test

Control: 1% Albumin Positive Result: INTENSE YELLOW coloration

Xanthoproteic Test
o Proteins when treated with conc. nitric acid turn yellow to orange when neutralized with sodium hydroxide. This is due to the nitration of the benzene ring present in tyrosine, tryptophan, and phenylalanine.

Xanthoproteic Test

Xanthoproteic test is used to identify the presence of a benzene ring so a positive result will change the solution color to yellow. In our experiment, sediments 1, 3 and the supernate yielded a positive while the sediment 2 had a lighter color so it yielded to a negative result. Protein is present in the nucleus, endoplasmic reticulum and the cytosol.

Test for Lipids

Sudan Test Acrolein Test

Sudan Test

Control: 1% Lecithin Positive Result: RED coloration

Sudan Test
o Sudan red is a fat-soluble dye that stains lipids red. Using Sudan red can show the amount and the location of lipids

Sudan Test

Sudan series of dyes do not react chemically with lipids while the dye molecules have a hydrophobic effect. All of our sediments yielded a positive result since its color is almost the same with the control solution.

Acrolein Test

Control: 1% Lecithin Positive Result: BURNT GREASE ODOR

Acrolein Test
o Lipids when heated with potassium bisulfate will release acrolein. Glycerol portion is dehydrated to form unsaturated aldehyde (acrolein). o Acrolein test is a test for the presence of glycerin or fats. A sample is heated with potassium bisulfate, and acrolein is released if the test is positive. When a fat is heated strongly in the presence of a dehydrating agent such as potassium bisulfate (KHSO4), the glycerol portion of the molecule is dehydrated to form the unsaturated aldehyde, acrolein (CH2=CHCHO), which has the peculiar odor of burnt grease.

Acrolein Test

Acrolein test is utilized to determine the presence of glycerin in a fat and can easily be detected by its odor. In our experiments, sediment 1 and 2 yielded positive results because it gave off a faint odor that is similar to that of the controls odor; while sediment 3 and the supernate gave a negative result. Glycerin is therefore present in the nucleus and mitochondria.

DATA & RESULTS

TEST
Carbohydrates a. Molisch b. Benedict Proteins a. Biuret b. Xanthoproteic

CONTROL SEDIMENT SEDIMENT SEDIMENT

1 1 % Ribose Purple ring Brick red ppt. 1% Albumin Purple solution Intense yellow solution 1% Lecithin Red coloration Biting odor
+++ ++ ++++

SUPERNATANT 3

++++

+ +++

+++

Lipids a. Sudan b. Acrolein

++++

++++

++++

+++++

++

CONCLUSION
It can be concluded that fractionation is a separation process in which a certain quantity of a mixture like solid, liquid, solute, suspension or isotope is divided up in a number of smaller quantities (fractions) in which the composition changes according to a gradient. Fractions are collected based on differences in a specific property of the individual components. After a series of centrifugation the first sediment yields nuclear fraction (containing both nuclei and unruptured cells). Sediment two is composed of mitochondrial fraction (containing mitochondria, lysosomes, peroxisomes). While in sediment three, a mixture of free ribosomes and smooth and rough endoplasmic reticulum was found. Lastly, the supernate 3 corresponds approximately to the cytosol of the cell. Therefore, by various modifications of this basic approach, it is generally possible to isolate each cell organelle in relatively pure form and the presence of each organelle is supported by the chemical or molecules present in the solution.

REFERENCES
Laboratory Guide in Biochemistry by Sylianco Biochemistry by Cabatit http://www.biosci.ohiou.edu/introbioslab/Bios170/170_2/benedict.htm http://en.wikipedia.org/wiki/Acrolein#Acrolein_test http://www.scribd.com/doc/91727176/Biochemistry-Postlab-Midterm