Biochemistry Postlab Midterm

BIOCHEMISTRYLABORATORY(Midterm)

Geromil J. Lara, RMT, MSMT

ACTIVITY 1A /1BSubcellular Components of the Living Cell

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

ACTIVITY 1A /1BSubcellular components of the Living Cell

• Sediment 1– Nuclear Fraction– Nuclei and Unbroken Cells

• Sediment 2– Mitochondria

• Sediment 3– Microsome– Proteins, Enzymes, Inorganic Ions


• Qualitative Tests– Carbohydrates

• Molisch Test• Benedict’s Test

– Proteins• Biuret Test• Xanthoproteic Test

– Lipids• Sudan Test• Acrolein Test


• Carbohydrates – Molisch Test

5 drops of the supernate + Molisch Reagent shake

Layer with 1 mL conc. H2SO4

Control: 1% LecithinMolisch Reagent: α-Naphthol dissolved in Ethanol

ACTIVITY 1A Subcellular components of the Living Cell




Principle: when sugar solution is mixed with alpha-naphthol 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




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


• Carbohydrates – Benedict’s Test

1 mL of Benedict’s Reagent + 2 drops of supernate

Boil for 2 minutes and allow to cool

Benedict’s Reagent: Sodium Citrate, Sodium Carbonate, Sodium Hydroxide, and Copper Sulfate

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

• Carbohydrates – Benedict’s 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

Benedict’s Test

CuSO4 + 2NaOH Cu(OH)2 + Na2SO4

2Cu(OH)2 Cu2O + 2H2O (Brick Red Precipitate)

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

Red precipitate = (+)


• Proteins – Biuret Test

5 drops of the supernate + 5 drops 10% NaOH+

0.5% CuSO4

Control: 1% AlbuminBenedict’s Reagent: Sodium Hydroxide and Copper

Sulfate


• 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


• 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


• 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


• 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 2AQualitative Tests for Proteins

• Millon’s Test

1 mL egg albumin + 2 drops Millon’s reagent mix and heat

(+) flesh precipitate to red color

• Millon’s 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


• 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


• 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 + 2 ninhydrin ---> CO2 + aldehyde + final complex(BLUE) + 4H2O

alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O alpha-amino acid + H2O ---> alpha-keto acid +NH3 alpha-keto acid + NH3 ---> aldehyde + CO2


• 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 2BPrecipitation 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


• 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


• 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


• Alcohols– 95% Alcohol (2 tubes)

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


• 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

• Millon’s Test, Xanthoproteic Test, Biuret Test, Hopkins Cole Test

ACTIVITY 3A: ENZYMES

• Potato Extract

– With starch and proteins– Rich in catalase and peroxidases


• Biuret Test

– Expected to have positive result

– Hydrolysis of peptides


• 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


• Test for Catalase Activity


• Test for Specificity of Enzyme Action

1 mL 1 mL Starch 1%

Glycogen + +

1 mL salivary AMS 1 mL salivary AMS

Stand for 15 mins. at RT and perform iodine test

1 2

1 Drop 1 DropSolution 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


• 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


• 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


• Effect of pH

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


• 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


• Effect of pH

TT 1: 2% pepsin + 0.4% HCl = AcidTT 2: 2% pepsin + 0.4% Na2CO3 = BasicTT 3: 2% pancreatin + 0.4% HCl = AcidTT 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


• Orcinol Test for Pentoses (Bial’s Test)

Dilute 0.5 mL sample with 2.5 mL water add

3 mL Orcinol Reagent heat

Blue Color


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


• Diphenylamine Test for Deoxyribose

1.5 mL sample + 3.5 mL diphenylamine

heat 10 minutes

green-blue color


• 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


• Test for Phosphates

2 mL sample + 2 mL Nitric Acid

add 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


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


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


ACROLEIN FORMATION

- 2H2O Glycerol Acrolein (acrid

odor) dehydrating agent (potassium bisulfate)


EMULSIFICATION OF FATS


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


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


SAPONIFICATION OF LARD– Sodium Carbonate – to produce hard soap

Palmitin Palmitate + Glycerol Soap


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


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)


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

Biochemistry Postlab Midterm

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