(3) METHODS FOR TP

Question 1

• Classical method for protein quantitation
• Reference method but not routinely used:
  o Time-consuming
  o Only an assumption so it is an inaccurate method

Answer 1

Kjeldahl method

Question 2

Principle: Measures the amount of Nitrogen in spx

Answer 2

Kjeldahl method

Question 3

3 steps of Kjeldahl method

Answer 3

Digestion → Distillation → Titration

Question 4

• Assumes that nitrogen content of protein is only 16% when it actually varies between 15.1-16.8%
• 1g of nitrogen = 6.54g of protein

Answer 4

Kjeldahl method

Question 5

• Most widely used method for TP determination
• Colorimetric/Non-enzymatic method

Answer 5

Biuret method

Question 6

Absorbance of color is read @ 540nm
(deep purple/violet)

Answer 6

Biuret method

Question 7

Principle: Based on # of peptide bonds present in protein forms a bond w/ cupric ion forming a violet-colored chelate.

Answer 7

Biuret method

o The darker the color, the more protein is present.
o How? Cu in alkaline CuSO4 will bind to peptide bonds present in protein forming a peptide-copper complex giving a violet-colored product.

Question 8

Biuret method Interference:

Answer 8

Lipemic sample

Question 9

Biuret method Reagents

Answer 9

o NaK tartrate (Rochelle salt): prevents ppt of Cu
o NaOH
o Alkaline CuSO4
o KI: acts as stabilizer

Question 10

• Principle: Oxidation of phenolic AA compounds such as tyrosine, tryptophan, & histidine.
• Has the highest analytical sensitivity (can detect very small amounts of protein)

Answer 10

Folin-Ciocalteu (LOWRY) method

Question 11

Folin-Ciocalteu method main reagent:

Answer 11

Phosphotungstic-molybdic acid or phenol rgt

o Can oxidize phenolic AA causing reduction of phosphotungstic-molybdic acid → deep blue color measured spectrophotometrically.

Question 12

Folin-Ciocalteu method color enhancer:

Answer 12

Biuret rgt

Question 13

• Principle: The absorbance of proteins at 210nm is due to the absorbance of the peptide bonds at specific wavelength.

Answer 13

UV ABSORPTION method

Question 14

UV absorption @ 280nm:

Answer 14

Aromatic AA such as tryptophan, tyrosine, phenylalanine

Question 15

• Based on measurement of refractive Index of serum TP
• Based on how protein bend light.
• Changes is proportional to conc. of proteins in sample.

Answer 15

Refractometry method

Question 16

Measurement depends on formation of a uniform fine precipitate which scatters incident light in suspension (nephelometry) or block light (turbidimetry).

Answer 16

TURBIDIMETRY & NEPHELOMETRY method

Question 17

• Dye-binding technique
• Used for detection of proteins as little as 1ug.

Answer 17

Coomassie Brilliant Blue dye method

Question 18

Develops violet color by reacting with primary amines widely used for detection of peptides & amino acids after paper chromatography.

Answer 18

Ninhydrin method

Question 19

Principle: Separation is based on migration of charged particles in an electric field

Answer 19

Serum Protein Electrophoresis (SPE) method

Question 20

2 sites of Serum Protein Electrophoresis (SPE) method

Answer 20

o (-) electrode: cathode
o (+) electrode: anode

Question 21

Isoelectric Property of Proteins

Answer 21

o Amphoteric: either positive or negative depending on pH condition
o No charge @ isoelectric point
✓ Isoelectric pt.: Net charge of proteins is 0.
o Acidic & basic AA content of proteins determines its net charge.

Question 22

Proteins move towards the anode bcos proteins are amphoteric.

Answer 22

SPE method

Question 23

Buffer for SPE:

Answer 23

Barbital (Veronal) – pH 8.6 (added to make proteins a (-) charge so it will migrate to (+) charge anode.

o ↑pH (basic): (-) charge proteins
o ↓pH (acidic): (+) charge proteins

Question 24

Normal SPE pattern:

Answer 24

(separates protein into 5 fractions)
From most anodal to least anodal:

1. Albumin band:
   o 1st band/most abundant/most anodal
2. a1 globulin band:
   o 2nd fastest band & a1-antitrypsin (AAT) is the major contributor in a1 globulin (90%).
3. a2 globulin:
4. B globulin band
5. y globulin band:
   o includes the immunoglobulins & least anodal

Question 25

major contributor in a1 globulin (90%).

Answer 25

a1-antitrypsin (AAT)

Question 26

NOTE!

Answer 26

! In SPE, pH is adjusted so that the electric charge of protein is (-) ! In Dye binding, protein is charged (+) to bind w/ the anionic dyes ((-) charge).