BCM EXPERIMENTS-1-4

protein

known to contain elements of carbon, hydrogen, oxygen, nitrogphosphorus together w/ traces of iron, copper, iodine, manganese

most abundant elements of proteins

carbon, hydrogen, oxygen, nitrogen

casein

C38H57N909

casein

main protein of milk

charring

indicates presence of carbon and removal of hydrogen & oxygen from casein

charring

incomplete combustion of certain solids when subjected to high heat

char

residual black carbon material from heating casein

moisture

indicates presence of hydrogen; liquid diffused/ condensed in relatively small quantity

heat distillation

removes water vapor and volatile organic compounds from the matrix

arg-tyr-leu-gly-tyr-leu

group of amino acids contained in casein (C38H57N9O9)

presence of nitrogen

can be tested in casein through mixture with soda lime (CaHNaO2) and heat

ammonia gas (NH3)

confirmatory product from heating casein (C38H57N9O9) with soda lime (CaHNaO2)

soda lime

used to absorb mixture and gases in the Nitrogen test

soda lime

mixture of calcium oxide (CaO) & sodium hydroxide (NaOH)

NH3 gas

turns red litmus paper to blue

positive test for the presence of nitrogen

urine-like odor & turning red litmus paper to blue

fusion mixture

2 parts sodium carbonate (Na2CO3), 1 part potassium nitrate (KNO3)

fusion

procedure that changed sulfur and phosphorus compounds into sulfate & phospate ions

fusion

used to oxidize sulfur to sulfate & phosphorus to phosphate

clear or white

resulting color of mixture when fusion mixture is added with casein when subjected to high heat

sulfur

its presence was tested by using one portion of filtrate from the fusion method with HCl and BaCl2

BaSO4

white ppt. that is formed when fusion mixture filtrate is added with HCl and BaCl2

hydrochloric acid

a reagent used to acidify filtrate from fusion mixture for the S test

BaCl2

a reagent used to create a white ppt. to test the presence of Sulfur

phosphorus

presence of this was tested using filtrate of fusion mixture, ammonium molybdate, and nitric acid to form a yellow ppt.

ammonium phosphomolybdate ppt

positive result for the presence of phosphorus

phosphorus test

A test that is used to determine the presence of phosphate groups in solutions

nitric acid (HNO3)

reagent used to acidify filtrate of fusion mixture for phosphorus test

ammonium molybdate (NH4)2MoO4

reagent used to create the yellow ppt. to test presence of phosphorus

biuret test

general test for proteins

violet-colored/ pink colored solution

This indicates a positive test for Biuret reaction

violet-colored solution

indicates presence of long chains of proteins

pink-colored solution

indicates presence of short chains of proteins

-CONH2

any compound containing this functional group is will also give a positive test for biuret test

biuret test

used to test the extent of protein hydrolysis through color reactions of violet, pink, or none at all

dark violet -colored solution

albumin + 10% NaOH + 0.5% CuSO4 =

peptide linkage

chemical structure in proteins responsible for a postive biuret test

will simple amino acids give positive biuret test

no

pink-colored solution

peptone solution + 10% NaOH + 0.5 CuSO4

Xanthoproteic Reaction

a reaction due to the presence of a phenyl group (-C6H5) in the protein molecule

xanthoproteic reaction

involves the nitration of phenyl rings present in the aromatic amino acids such as tyrosine, phenylalanine, and tryptophan forming yellow nitro-substitution products that turn orange when an alkali is added (salt formation)

yellow-colored solution

albumin + conc. nitric acid + heat =

orange-colored solution

albumin + conc. nitric acid + heat + ammonium hydroxide =

aromatic amino acids

what kinds of amino acids will be detected using the xanthoproteic reaction

nitration

process of introducing a nitro group into an organic chemical compound

ammonium hydroxide (NH4OH)

alkali used to neutralize protein solutions like albumin with an acidic pH (HNO3) turning orange

tyrosine, trytophan

common amino acids that undergo nitration pro

yellow

color of albumin solution when added with conc. nitric acid

orange

color of albumin solution when added with conc. nitric acid and ammonium hydroxide

millon’s reagent

reagent that is prepared by mixing 1 part by weight of mercury with two parts of weight by conc. nitric acid and diluted with water

mercuric nitrate (Hg(NO3)2)

millon’s reagent is otherwise known as this

red flocculent ppt.

albumin sol’n + millon’s reagent =

phenol group of tyrosine

responsible for the formation of a red flocculent ppt. when Millon’s reagent is added to albumin

tyrosine (C9H11NO3)

amino acid that gives a positive Millon’s test

Millon’s test

A test used to detect the amino acid tyrosine containing the phenol group

Hopkins-Cole reagent

Prepared by mixing 10 g of powdered magnesium with cold water and then filtered. Filtrate obtained is then acidified with acetic acid to prevent partial precipitation of magnesium

violet ring formation

When albumin (protein) is mixed with glyoxylic acid (CHOCOOH) and is treated with sulfuric acid (H2SO4)

proteins containing tryptophan

what causes a positive Glyoxylic acid reaction

indole group of trytophan

what reacts with glyoxylic acid in the presence of conc. H2SO4 to give a purple ring

glyoxylic acid

reagent that is prepared by reducing oxalic acid with magnesium powder or sodium amalgam

glacial acetic acid

a solution that contains glyoxylic acid when exposed to sunlight

Hopkins-Cole test

a test that is specific to tryptophan or an amino acid containing an indole group

indole group

a functional group that reacts with glyoxylic acid in the presence of a strong acid (H2SO4) to form a violet cyclic product

Heller’s ring test

a test used to clinically detect the presence of albumin in urine

albumin sol’n + conc. nitric acid =

formation of a white ring or ppt at the junction of two solutions

conc. nitric acid

what causes denaturation of proteins in the Heller’s ring test

denatured

when proteins are treated with strong mineral acids, they…

albumin + 40% NaOH + lead acetate sol’n =

black ppt. (lead sulfide)

sodium sulfide (Na2S)

loosely combined sulfur after boiling with sodium hydroxide forms

black-colored ppt

Na2S + lead acetate forms what color of ppt.

black deposit of lead sulfide (PbS) in albumin

indication of a positive reduced sulfur test

cysteine, methionine

kinds of amino acid that contain reduced sulfur group

Adamkiewicz reaction

a test that gives a positive result with amino acids containing indole derivatives (tryptophan)

albumin + glacial acetic acid + conc. sulfuric acid =

near isoelectric point

when does heat coagulation and precipitation occur best

albumin

protein that coagulates when heated

aqueous solutions of proteoses, peptones, gelatine, and casein

protein solutions that don’t coagulate by heat

no coagulation occurred

2% casein +heat =

pH 4.6

at what pH level do casein proteins denature to coagulate

albumin denatures

albumin + 0.1 N acetic acid + heat=

denatures albumin but also dissolves ppt

albumin + 0.5 N acetic acid + heat =

isoelectric point

the pH where the molecule has no net charge

acids and bases

alters pH therefore changes the isoelectric point

denaturation

process where proteins or nucleic acids lose quaternary, tertiary, secondary structures because of the presence of stress: acid, base, salt, or heat

1 N acetic acid

acetic acid concentration best for albumin coagulation to occur

normality (N)

A measure of concentration equal to the gram equivalent weight per liter of solution

white ppt.

ethyl alcohol + 2% albumin + water =

protein has been completely precipitated by alcohol

what happens when no coagulation occurs

alcohol

disrupts the intrahydrogen bonds of proteins causing shrinkage and hardenning of tissues for preservation processes

red ppt

solid ammonium sulfate & albumin ppt. + Millon’s reagent =

blue-colored solution

solid ammonium sulfate & albumin filtrate + NaOH + CuSO4 =

pink-colored solution

solid ammonium sulfate & 10% commercial peptone + NaOH + CuSO4 =

Salting out of proteins is successfully done and albumin does not contain peptide bonds anymore

(NH4)2SO4 & albumin filtrate in Biuret test turns to a blue-colored solution indicates

addition of salts and electrolytes

leads to the absorption of a solution envelope from colloidal particles of a protein solution along with the neutralization of surface charges leading the protein to precipitate

ammonium sulfate, magnesium sulfate

neutral salts that are used to “salt out” proteins from protein solutions

salting out

purification method that utilizes the reduced solubility of molecules in a solution of high ionic strength (saturation)

Salting out

causes reduction of molecules solubility

white ppt

albumin + Pb(Ac)2 =