Analysis and Denaturation of Proteins Flashcards
function as biological catalysts or enzymes, transporter of oxygen and hormone
proteins
repeating unit of protein
amino acid
4 levels of protein structure
- Primary structure
- Secondary structure
- Tertiary structure
- Quaternary structure
composed of single covalently bonded amino acid
primary structure
two types of secondary structure
alpha helix structure
beta-sheet structure
contains one strand of amino acid chain that is bonded by intramolecular hydrogen bond
alpha helix structure
two chains that are linked by hydrogen bonds
beta-sheet structure
combination of pure helix or pure beta, or a combination of both
tertiary structure
stabilizing interactions in tertiary level
- salt linkages
- hydrophobic interactions
- disulfide linkages
- hydrogen bonds
interaction of two or more folded polypeptides
quaternary structure
is the unfolding of complex secondary, tertiary, and quaternary structure of protein
denaturation
what can denature proteins?
- heat
- strong acid
- organic solvents/acids
- heavy metals
it causes the atom within the protein molecule to vibrate more rapidly, causing the hydrogen bonds and hydrophobic interactions to break.
heat
they split salt linkages by ionizing the caboxylic group
strong acids
it denatures the protein by disrupting the hydrogen bonds
alcohol
they denature proteins by combining the carboxylate anions of the acidic amino acid with the metal, causing precipitation
heavy metal ions (silver, lead, mercury)
are used to precipitate alkaloids, giving “alkaloidal reagents”
organic acids like picric acid and tannic acid
the anion of these acids will react to the protonated amino acid group of protein, causing the disruption of salt linkages
organic acids ( picric acid and tannic acid)
Color Tests of Protein
- Biuret Test (2 mL 10% NaOH + 5 drops CuSO4)
- Ninhydrin Test
- Xanthoproteic Test
- Millon’s Test
- Hopkin’s Cole Test
- Sakaguchi Test
- Lead Acetate Test (Test for Sulfur)
Positive result for compounds that contain two or more peptide linkages
Biuret Test
Color of biuret test
purple (due to complex by cupric ions)
dipeptides and amino acids that do not give positive result to BT
serine, threonine
when amino acids are sprayed w/ ninhydrin, it will give
blue to violet
the presence of amino group including amine and ammonia will give positive result in NT except
proline & hydroxyproline (yellow)
in XT, nitration of amino acids that contain benzene ring will yield the product of ____ that will give color
nitrobenze, yellow to orange
in XT, they will produce nitrobenzene when treated w/ concentrated nitric acid
- tryptophan
- phenylalanine
- tyrosine
in XT, what do not give a positive reaction
collagen and gelatin
in MT, the phenol group in amino acid like tyrosine is nitrated by a solution of ___ in concentrated nitric acid
mercuric and mercurous nitrates
what will be formed in millon’s test
white precipitate, turning brick red (due to mercury complex)
in MT, addition of ____ will turn the precipitate to
NaNO2, darker pink or red
color of precipitate in MT
white - brick red (due to mercury complex - darker pink/red (upon addition of NaNO2
BT is for
two or more peptide linkages
XT is for
amino acids that contain benzene ring
Ninhydrin is for
amino group
MT is for
phenol group (ex. tyrosine)
HT is for
aldehyde
in HT, the aldehyde present in reagent will cause the formation of a
blue or violet colored ring (due to formation of a complex bet. reagent and indole ring
ST is to identify the presence of
arginine
in ST, sodium hypobromite or hypochlorite react with guanido group giving a
red-orange colored complex
This is a test for sulfur-containing amino acids like cysteine and methionine
Lead Acetate Test
The sulfahydryl or disulfide group are converted to
Na2S (inorganic sulfide)
Na2S reacts with lead acetate to form
brownish-black precipitate of lead sulfide (Pbs)
Xanthoproteic Procedure
reagents: HNO3, NH4OH
albumin - yellow, richer yellow
gelatin - no reaction
casein - pale yellow, richer yellow
Millon’s Procedure
reagents: millon’s reagent, 0.1% NaOH
albumin - red, yellow-orange
gelatin - no reaction
casein - red, yellow-orange
HCT Procedure
reagents: HCT reagent, sulfuric acid
albumin - blue ring below the albumin
gelatin - no reaction
casein - blue ring below the milk
Lead Acetate Procedure
reagents: 5 drops 10% NaOH, 3 drops 5% Lead acetate
albumin - black precipitate (Pbs)
gelatin - no precipitate
NT Procedure
reagents: Ninhydrin
albumin - dark violet top, white bottom
gelatin - no reaction
casein - violet
Biuret Procedure
reagents: 10% NaOH, 5 drops CuSO4
albumin - slightly cloudy, dark purple
gelatin - orange to red
casein - purple
Heat
albumin + 3 mL water + boiling water bath:
after cooling = cloudy
color: purple/violet (peptide bond)
Heat
albumin + 3 mL water
- violet
Inorganic Acids
3 mL 5% albumin + HCL = precipitate at 8th drop
3 mL 5% albumin + H2SO4 = precipitate at 8th drop, increased 2 drops
Alcohol
reagents: 5 mL 95% ethanol
albumin - no precipitate, albumin became opaque
gelatin - no precipitate formed
casein - white precipitate
Heavy Metals
reagents: egg albumin
lead acetate - black precipitate
silver nitrate - white precipitate mercuric chloride - cloudy or white
