PROTEIN DETERMINATION Flashcards
determination of amino Nitrogen derived from protein
KJELDAHL METHOD
Functional groups:
amino group and carboxyl group
KJELDAHL METHOD
Nitrogen converted to [?] using [?]
NH3 [ammonium]
H2SO4 [bisulfate]
Protein + H2SO4 ——> NH4 + H2SO4
KJELDAHL METHOD
(NH4) ——> Na2SO4 + NH3 + 2H2O
KJELDAHL METHOD
KJELDAHL METHOD
The nitrogen in ammonia may be measured using:
Nesslerization
Berthelot reaction
Titration method
- basis for measuring the amount of protein] HSO4 + 2NaOH [sodium hydroxide
ammonia
ammonia reacts with Nessler’s reagent or double iodide salt of potassium and mercury
Nesslerization
Urea and ammonia determination
Nesslerization
Berthelot reaction
ammonia reacts with phenol/phenolic cpd and a hypochlorite
Berthelot reaction
Berthelot reaction End product:
Indophenol
Copper binds to the peptide bond
BIURET METHOD
(+) purple-color
BIURET METHOD
= purple chromogen
Protein + CuSO4 + NaOH + Rochelle salt
Employed spectrophotometric methods
BIURET METHOD
Tyrosine & tryptophan reduce PT-PMA reagent = blue color
FOLIN-CIOCALTEU METHOD
Detects proteins in low conc. (10-60 pig/ )
FOLIN-CIOCALTEU METHOD
Widely used in research to measure tissue & enzyme proteins
FOLIN-CIOCALTEU METHOD
Not used in routine laboratory work
FOLIN-CIOCALTEU METHOD
Notsatisfactory for urine & CSF protein determination
FOLIN-CIOCALTEU METHOD
PT-PMA
phosphotungstic phosphomolybdic acid
Dueto the high electron density of aromatic rings (benzene) of tyrosine & tryptophan in solution (pH 8)
ABSORPTION OF UV LIGHT AT 280 NM
- anionic dye where albumin binds itself
BromcresolGreen Method (BCG)
Albumin at pH [?] (acidic) is [?] (positive charge) & binds w/ [?]
4.2
cationic
anionic dye
Dye-binding Methods used in spectrophotometric determination of serum albumin
(Bromcresol Green or Bromcresol purple)
DYE-BINDING METHODS
Other dyes:
Bromcresol purple
Methyl orange
HABA(2 4-dihydroxyazobenzenebenzoic acid)
PSP (Phenolsulfonphthalein
Detection of protein fractions after electrophoresis
DYE-BINDING METHODS
TURBIDIMETRY & NEPHELOMETRY
Precipitating agents
Sulfosalicylic acid (SSA) with sodium sulfate
Trichloroacetic acid
- to facilitate precipitation of both albumin and globulins
Sulfosalicylic acid (SSA) with sodium sulfate
are often used for measuring urine & CSF protein
Turbidimetric methods
[?] pH: protein donates protons = [?] charge
Alkaline
(-)
Migrates towards ANODE
AlkalinepH: protein donates protons = (-) charge
Acidic pH
(+) charge
Migrates towards CATHODE
Acidic pH: protein it receives protons = (+) charge
Typicalfor serum electrophoresis
Migrates towards ANODE
No fibrinogen (consumed during coagulation)
Migrates towards ANODE
No fibrinogen
AlkalinepH: protein donates protons = (-) charge
migrates the fastest toward the anode due to its low molecular
weight
Albumin
Most anodal
Albumin
Highest peak (most abundant protein in plasma)
Albumin
The [?] manifest the concentration of the protein present (directly proportional)
peak of protein fraction
Ex. Gamma globulin
Acidic pH: protein it receives protons = (+) charge
Ex. Albumin
Alkaline pH: protein donates protons = (-) charge
Protein stains after electrophoresis
Coomassie Brilliant Blue
Nigrosin dye
Ponceau S dye
causes gamma globulins to migrate toward the cathode even though they are slightly (-) charged
ELECTROENDOSMOSIS
due to electrical charge on support medium
ELECTROENDOSMOSIS
Influences the migration of gamma globulins towards the cathode
electrical charge on support medium
Relative concentration of each band determined by
densitometry
plasma samples result in fibrinogen peak between [?]
gamma and beta fractions
: due to IgA; Cirrhosis
Beta-gamma bridging effect (in serum)
: due to Fibrinogen
Beta-gamma bridging effect (in plasma)
: Monoclonal gammopathy (Multiple myeloma)
Monoclonal band (gamma globulin)
: Chronic inflammation
Polyclonal band
: nephrotic syndrome
Increase in a-2-macroglobulin
: deficiency in a-1-antitrypsin; Juvenile cirrhosis
a-1-globulin flat curve
