BIOCHEMISTRY LABORATORY (Midterm)

Geromil J. Lara, RMT, MSMT

ACTIVITY 1A /1B Subcellular Components of the Living Cell

5% Trichloroacetic Acid
Suspending medium Able to precipitate proteins Good fixative and preservative

ACTIVITY 1A /1B Subcellular components of the Living Cell

Sediment 1
Nuclear Fraction Nuclei and Unbroken Cells

Sediment 2
Mitochondria

Sediment 3
Microsome Proteins, Enzymes, Inorganic Ions

ACTIVITY 1A /1B Subcellular components of the Living Cell

Qualitative Tests
Carbohydrates
Molisch Test Benedicts Test

Proteins
Biuret Test Xanthoproteic Test

Lipids
Sudan Test Acrolein Test

ACTIVITY 1A /1B Subcellular Components of the Living Cell

Carbohydrates Molisch Test

5 drops of the supernate + Molisch Reagent shake Layer with 1 mL conc. H2SO4

Control: 1% Lecithin Molisch Reagent: -Naphthol dissolved in Ethanol

ACTIVITY 1A Subcellular components of the Living Cell

Carbohydrates Molisch Test

5 drops of the supernate + Molisch Reagent shake Layer with 1 mL conc. H2SO4

Principle: when sugar solution is mixed with alphanaphthol is brought in contact with conc. H2SO4, a violet ring is formed at the junction of the 2 liquids

ACTIVITY 1A Subcellular components of the Living Cell

Carbohydrates Molisch Test

5 drops of the supernate + Molisch Reagent shake Layer with 1 mL conc. H2SO4

H2SO4 : acts as dehydrating agent forming furfural derivatives which interact with alpha-naphthol liberating a colored compound

ACTIVITY 1A /1B Subcellular Components of the Living Cell

Carbohydrates Benedicts Test

1 mL of Benedicts Reagent + 2 drops of supernate

Boil for 2 minutes and allow to cool

Benedicts Reagent: Sodium Citrate, Sodium Carbonate, Sodium Hydroxide, and Copper Sulfate

ACTIVITY 1A/1B Subcellular components of the Living Cell

Carbohydrates Benedicts Test

used to detect the presence of all monosaccharides, and generally all reducing sugars Will detect the presence of aldehydes (except aromatic ones), and alpha-hydroxy-ketones, including those that occur in certain ketoses

Benedicts Test
CuSO4 + 2NaOH 2Cu(OH)2 Cu(OH)2 + Na2SO4 Cu2O + 2H2O (Brick Red Precipitate)

Blue = (-) Green, Yellow, Orange, Red, & then Brick Red precipitate = (+)

ACTIVITY 1A /1B Subcellular components of the Living Cell

Proteins Biuret Test

5 drops of the supernate + 5 drops 10% NaOH + 0.5% CuSO4 Control: 1% Albumin Benedicts Reagent: Sodium Hydroxide and Copper Sulfate

ACTIVITY 1A /1B Subcellular Components of the Living Cell

Proteins Biuret Test

Alkaline solution of proteins treated with copper sulfate results in the production of a rose-pink to violet, then purple Test used to detect the presence of peptide bonds by hydrolysis

(+) 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

ACTIVITY 1A /1B Subcellular Components of the Living Cell

Proteins Xanthoproteic Test

1 mL of the supernate + 1 mL conc. HNO3 heat add 5 drops NH4OH Proteins when treated with conc. nitric acid turn yellow to orange when neutralized with sodium hydroxide Due to the nitration of the benzene ring present in tyrosine, tryptophan, and phenylalanine

Tyrosine

Tryptophan

Phenylalanine

ACTIVITY 1A /1B Subcellular components of the Living Cell

Lipids Sudan Test

5 drops of the supernate + 5 drops of Sudan IV (Red)

Control: 1% Lecithin Sudan IV: fat-soluble dye used for the staining of lipids, triglycerides, and lipoproteins

ACTIVITY 1A /1B Subcellular Components of the Living Cell

Lipids Acrolein Test

0.5 g of KHSO4 + a drop of the supernate heat odor of burnt grease Lipids when heated with potassium bisulfate will release acrolein Glycerol portion is dehydrated to form unsaturated aldehyde (acrolein)

ACTIVITY 2A Qualitative Tests for Proteins

Millons Test
1 mL egg albumin + 2 drops Millons reagent mix and heat (+) flesh precipitate to red color Millons reagent: mercurous nitrate in nitric acid Protein is precipitated as mercury salt and after heating precipitated turns flesh to red color Due to the phenol group contained in tyrosine

ACTIVITY 2A Qualitative Tests for Proteins

Glyoxylic Acid reaction (Hopkins-Cole Test)

1 mL egg albumin + 1 mL Hopkins-Cole reagent

allow 10 drops pure conc. H2SO4 When protein mixed with glyoxylic acid and H2SO4, a violet ring will form beneath the protein mixture Due to the presence of an indole nucleus in the tryptophan component Tryptophan condenses with the aldehyde to form the colored product

ACTIVITY 2A Qualitative Tests for Proteins

Ninhydrin Test
1 mL egg albumin + 1 mL 0.1% Ninhydrin Reagent heat to boiling blue product
When protein is boiled with ninhydrin, a blue color is produced Due to the presence of the alpha-amino acid All proteins are positive except proline

alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O alpha-amino acid + H2O ---> alpha-keto acid +NH3 alpha-keto acid + NH3 ---> aldehyde + CO2
alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BLUE) + 4H2O

ACTIVITY 2A Qualitative Tests for Proteins

Sakaguchi Reaction for Arginine

1 mL egg albumin + 1 mL 10% NaOH add 6 drops dil. alc. alpha-naphthol + 10 drops Na hypochlorite
for the detection of a specific type of protein with the amino acid containing the guanidinium group to form a red color Due to the presence of arginine

ACTIVITY 2B Precipitation Test for Proteins

Concentrated Minerals and Organic Acids Concentrated H2SO4 Concentrated HCl Concentrated HNO3 Glacial CH3COOH Strong acids cause the carboxyl end of the protein to remain undissociated and all amino groups to become fully proteinated

ACTIVITY 2B Precipitation Test for Proteins

Metallic Salts Mercuric Chloride Lead Acetate Copper Sulfate Ferric Chloride Barium Chloride Positive charges of these cations counteract the negative charge of the carboxyl group giving a precipitate

ACTIVITY 2B Precipitation Test for Proteins

Alkaloidal Reagents Picric Acid Trichloroacetic Acid Tannic Acid Phosphotungstic Acid Precipitation indicates the presence of both negative and positive charges and hence the ampotheric nature of proteins

ACTIVITY 2B Precipitation Test for Proteins

Alcohols 95% Alcohol (2 tubes)

1st tube: add diluted HCl 2nd tube: add 10% NaOH

ACTIVITY 2B Precipitation Test for Proteins

Coagulation by Heat Boiling the egg albumin Add 2 drops acetic acid Heat disrupts hydrogen bonds of secondary and tertiary structures while the primary structure remains unaffected

Millons Test, Xanthoproteic Test, Biuret Test, Hopkins Cole Test

ACTIVITY 3A: ENZYMES

Potato Extract
With starch and proteins Rich in catalase and peroxidases

ACTIVITY 3A: ENZYMES

Biuret Test
Expected to have positive result Hydrolysis of peptides

ACTIVITY 3A: ENZYMES

Test for Catalase Activity 5 mL of potato extract + 1 mL 3% H2O2 Observe for gas production Add 1 mL 0.5% benzidine Blue to Green

ACTIVITY 3A: ENZYMES

Test for Catalase Activity

ACTIVITY 3A: ENZYMES

Test for Specificity of Enzyme Action

1 mL Starch + 1 mL salivary AMS

1 mL 1% Glycogen + 1 mL salivary AMS

Stand for 15 mins. at RT and perform iodine test

1 Drop Solution

1 Drop Iodine

Iodine Test
Used for the presence of CHO Potassium iodide KI reacts with starch producing a deep purple color
Results of the formation of polyiodide chains

ACTIVITY 3A: ENZYMES

Iodine Test Amylose (straight chain)
Forms helices where iodide molecules assemble Blue-black color Starch

Amylopectin (branched portion)

Forms much shorter helices and iodine are unable to assemble orange/yellow hue Glycogen

ACTIVITY 3B: Factors Affecting Enzyme Action

Effect of Temperature 1% cooked starch + 1 mL saliva (amylase) Test Tube 1: 40 degrees Celsius Test Tube 2: 60 degrees Celsius Test Tube 3: 10 degrees Celsius Perform Iodine Test

ACTIVITY 3B: Factors Affecting Enzyme Action

Effect of pH Coagulated Egg White to 4 tubes Test Tube 1: 2% pepsin + 0.4% HCl Test Tube 2: 2% pepsin + 0.4% Na2CO3 Test Tube 3: 2% pancreatin + 0.4% HCl Test Tube 4: 2% pancreatin + 0.4% Na2CO3
Incubate at 40 degrees Celsius Determine the pH Perform Biuret Test
Control: 0.5% Peptone

ACTIVITY 3B: Factors Affecting Enzyme Action

Effect of pH

TT 1: 2% pepsin + 0.4% HCl = Acid TT 2: 2% pepsin + 0.4% Na2CO3 = Basic TT 3: 2% pancreatin + 0.4% HCl = Acid TT 4: 2% pancreatin + 0.4% Na2CO3 = Basic

ACTIVITY 3B: Factors Affecting Enzyme Action

Effect of pH
Pepsin Released by the chief cells in the stomach Optimum activity at pH 1-2 Degrades food proteins into peptides Pancreatin Produced by the exocrine cells of the pancreas With AMS, LPS, protease, and trypsin Optimum activity at pH 7-9

ACTIVITY 3B: Factors Affecting Enzyme Action

Effect of pH

TT 1: 2% pepsin + 0.4% HCl = Acid TT 2: 2% pepsin + 0.4% Na2CO3 = Basic TT 3: 2% pancreatin + 0.4% HCl = Acid TT 4: 2% pancreatin + 0.4% Na2CO3 = Basic

ACTIVITY 4: NUCLEIC ACIDS

Liver is suspended in 5% TCA To extract the acid soluble substance Residue is extracted with alcohol-ether To remove the lipids Residue is resuspended with 5% TCA To preserve the nucleic acid

ACTIVITY 4: Nucleic Acids

Test for Purine
1 mL of test solution + 1 mL 2N HCl Place in boiling water for 20 minutes to hydrolyze the nucleic acid to form free purines Add 1 mL 2N HCl and 2 mL acetate buffer Place in boiling water Add 0.5 mL 10% CuSO4 bluish-brown ppt Add 0.5 mL sat. Na bisulfate Heat and a white or light tan flocculent ppt

ACTIVITY 4: Nucleic Acids

Orcinol Test for Pentoses (Bials Test)
Dilute 0.5 mL sample with 2.5 mL water add 3 mL Orcinol Reagent heat Blue Color

ACTIVITY 4: NUCLEIC ACIDS

Orcinol Reagent HCl + FeCl3 Pentose is dehydrated to form furfural Furfural reacts with orcinol Iron will produce a bluish product

ACTIVITY 4: Nucleic Acids

Diphenylamine Test for Deoxyribose

1.5 mL sample + heat

3.5 mL diphenylamine 10 minutes

green-blue color

ACTIVITY 4: NUCLEIC ACIDS

Diphenylamine Test for Deoxyribose Reaction depends on the conversion of the pentose to w-hydroxylaevulinic aldehyde which then reacts with diphenylamine to give a blue colored complex

ACTIVITY 4: Nucleic Acids

Test for Phosphates

2 mL sample add

2 mL Nitric Acid ammonium molybdate

yellow phospho-ammonium molybdate

CHARACERIZATION OF LIPIDS
SOLUBILITY
Insoluble in water
Insoluble in ordinary solvents Readily dissolve in chloroform, benzene, ether, boiling alcohol and other organic solvents

CHARACERIZATION OF LIPIDS
FORMATION OF TRANSLUCENT SPOT
Lipids have a characteristic greasy feel When brought in contact with a substance like paper, penetrate through it producing a translucent spot
Fats are non-volatile In RT, the spot of water can absorb enough heat from the air and evaporized But the spot of grease can never absorb enough heat to evaporized When the liquid is inside the sheet of paper, it diffracts light TRANSLUCENT PHENOMENON

CHARACERIZATION OF LIPIDS
REACTION OF FATS
Fatty acids are carboxylic acids and are therefore weak acids For fatty acids, the value of pKa is around 4.5. Therefore, generally speaking, fatty acids are neutral below pH 4.5 and charged above pH 4.5 Fats containing high unsaturated fatty acids are neutral in reaction, but when exposed to air become acidic due to hydrolysis which results from the liberation of volatile fatty acids

CHARACERIZATION OF LIPIDS
ACROLEIN FORMATION - 2H2O Glycerol Acrolein (acrid odor) dehydrating agent (potassium bisulfate)

CHARACERIZATION OF LIPIDS
EMULSIFICATION OF FATS

CHARACERIZATION OF LIPIDS
EMULSION
Is a mixture of two or more materials that are ordinarily immiscible Droplets of the dispersed component rapidly coalesce to form a separate layer Emulsifying agent must be present to stabilize the emulsion
Lecithin in the egg will serve as emulsifier

CHARACERIZATION OF LIPIDS
SAPONIFICATION OF LARD
Alcoholic Potash
KOH dissolved in ethanol To neutralized fatty acids in the lard

A metallic salt of fatty acid is formed Hydrophobic tails extend into the greasy droplets whereas the polar heads of the soap molecules face toward the water

CHARACERIZATION OF LIPIDS
SAPONIFICATION OF LARD
Sodium Carbonate to produce hard soap

Palmitin

Palmitate + Glycerol Soap

CHARACERIZATION OF LIPIDS
SEPARATION OF CHOLESTEROL AND TRIGLYCERIDES
Precipitate = cholesterol digitonide after digitonin precipitation Supernatant = triglycerides PRECIPITATE tested for Salkowski Test and Liebermann-Burchardt Test
Test for cholesterol

CHARACERIZATION OF LIPIDS
SALKOWSKI TEST
The presence of a double bond in one cholesterol rings is responsible for its ability to form color products in the presence of concentrated inorganic acids Sulfuric acid
Results in dehydration of cholesterol molecule with a formation of a red bicholestadien disulphonate

Bluish color between the 1st layer (chloroform) and 2nd layer (H2SO4)

CHARACERIZATION OF LIPIDS
LIEBERMAN-BURCHARD TEST
Deep green color (+)
due to the hydroxyl group (-OH) of cholesterol reacting with the reagents (acetic anhydride and concentrated sulfuric acid) and increasing the conjugation of the un-saturation in the adjacent fused ring

GOOD LUCK