Protein Analysis

1
Protein Analysis
Chapter 9
2
Other Common Protein Methods
3
Biuret Method

• Cupric ions react with peptide bonds under
  alkaline conditions producing a violet-purplish
  color
• (copper sulfate K-Na-tartrate NaOH)
• Measure color in SPEC at 540 nm

4
Biuret Method
Mix sample with biuret reagent Let stand at room
temperature for 15-30 min Filter or centrifuge
before reading if mixture is not clear Read
absorbance at 540 nm Construct a standard curve
using bovine serum albumin
5

• BIURET METHOD
• Advantages
• - Cheaper and faster than Kjeldahl
• - Less problem with color deviations vs Lowry, UV
  
• - Few substances interfere
• - Does not measure non protein nitrogen (NPN)
• Disadvantages
• - not sensitive to 2-4 mg level
• - bile pigments interfere
• - ammonium salts interfere
• - color depends on protein
• - lipids and carbos can affect clarity of
  solution
• - PROTEIN MUST BE SOLUBLE

6
LOWRY METHOD

• The Lowry method is a colorimetric method based
  on the formation of a blue color formed
• Tyrosine and/or tryptophan in a protein reduces a
  phosphomolybdic-phosphotungstic reagent
  (Folin-Ciocalteu reagent) in the presence of
  K-Na-tartrate in alkali (Biuret reagent)
• Absorbance values are determined on a
  spectrophotometer at 750 nm. As little as 0.2 mg
  protein in a sample can be determined.

7
LOWRY METHOD-Combination of Biuret and
Folin-Ciocalteu reagent

• Cu2 is reduced to Cu in the presence of
  proteins at high pH the biuret reagent chelates
  the Cu ion, then the FolinCiocalteu reagent
  enhances the blue color via chemical reduction
• Careful addition of reagents and very careful
  timing is very important to this assay.

8
LOWRY METHOD- Advantages

• This is the most sensitive spectrophotometric
  method available for determining total protein.
  It is 10 to 20 times more sensitive than UV
  absorption at 280 nm (next topic)
• This method is more specific than most other
  protein methods and is better at handling
  problems from turbidity in protein solutions
• The Lowry method is relatively rapid, requiring
  1-2 hours for analysis
• Widely used in biomedical field

9
LOWRY METHOD- Disadvantages

• This method requires careful standardization
  (making a good standard curve) because
• a. The amount of color varies with different
  proteins.
• b. The color is not strictly proportional to the
  concentration, due to matrix effects
• c. Recent evidence suggests that sucrose, lipids,
  some buffers, monosaccharides and hexosamines
  react to varying degrees with the reagents
• d. High concentrations of ammonium sulfate,
  sulfhydryl compounds, and phosphate can interfere

10
UV Absorption (280 nm)

• Most proteins exhibit strong UV light absorption
  at 280 nm because they contain chromophoric
  side chains such as tyrosine, tryptophan, and
  phenylalanine.

11
Amino Acid Structures
PHENYLALANINE
TYROSINE
TRYPTOPHAN
12
UV Absorption (280 nm)
Assuming a reasonably constant level of these
amino acids in food proteins, the concentration
of protein in a non-turbid solution is
proportional to the absorbance When we talk of
absorbance and concentration in the same
sentence, what should this AUTOMATICALLY make
you think of?
13
UV Absorption (280 nm)

• Absorbance according to Beer's Law
• A abc
• Where A Absorbance
• a absorptivity, a constant that is
  characteristic of a particular chemical species
  at a particular wavelength.b path length in
  cmc concentration of the absorbing chemical
  species

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UV Absorption (280 nm)

• If we assume that the concentration is to be in
  units of Molarity (moles/Liter), then we can use
  the molar absorptivity (e) in place of the
  absorptivity. The equation would then be
• Aebc
• Molar absorptivity can be determined for
  individual proteins if they are pure (no other
  proteins present).
• This can then be used to estimate unknown
  concentrations of that particular protein

15
UV ABSORPTION (280 nm)

• Advantages
• - rapid and sensitive
• - nondestructive
• - no ammonium interference (used to isolate
  proteins)
• Disadvantages
• - nucleic and phenolic acids also absorb at 280
  nm
• - amounts of Trp and Tyr vary with protein types
• - turbidity (cloudiness in solution) is a problem
• Applications of method? Not widely accepted for
  general food analysis more useful for research
  purposes by monitoring the extraction or
  separation of proteins

16

• Coomassie Brilliant Blue solution will directly
  bind to specific amino acids and protein tertiary
  structures
• The dye's color changes from reddish-brown to
  blue
• Absorbance at 595 nm read.
• Pros
• Rapid assay
• Useful when accuracy is not crucial
• Cons
• High protein-to-protein variation
• Not compatible with detergents (used to isolate
  proteins)
• Applications Finds use as rapid protein method
  in food and biomedical industry.

17
Bradford protein assay - showing increasing
amounts of protein concentration
18
Ninhydrin

• Primary amino groups on the end of proteins,
  peptides, and free amino acids will react with
  ninhydrin.
• This reaction forms a strongly colored purple
  solution referred to as Ruheman's purple. Read at
  570 nm.
• Sample can be tested for the amount of primary
  amino acids currently present, or sample can be
  alkaline hydrolyzed to increase the amount of
  these amino acids.

Common method for cheese
19

• Ninhydrin
• Advantages are
• Faster and more convenient that Kjeldahl
• Disadvantages are
• Large dilutions are necessary for spec. reading.
• Proteins differ in the dye binding capacity
• Make standard curve based on predominant primary
  amino acid present in the food.
• NPN, calcium, or phosphorous constituents will
  bind to the dye or to protein, causing
  interference.
• Addition of a metal chelator (i.e.. oxalic acid)
  may help reduce binding.

20

• BICINCHONIC ACID (BCA) METHOD
• Proteins reduce cupric ions to cuprous ions under
  alkaline conditions. Cuprous ions react with BCA
  reagent to give a purple color
• Advantages
• - as sensitive as Lowry but simpler
• - reagent more stable than Lowry
• Disadvantages
• - color not stable with timeprecise timing
• - reducing sugars interfere more than Lowry
• - color variations between proteins occur
• absorbance vs concentration not absolutely linear

21
Dumas Method

• Measures the Nitrogen released upon combustion of
  the sample (800C). Measured by GC using a
  Thermal Conductivity Detector (TCD). May be used
  for labeling purposes.

22
Dumas Method
Alternative to Kjeldahl suitable for all types
of foods Advantages Requires no hazardous
chemicals Can be accomplished in 3
min. Automated instruments for up to 150
samples Disadvantages Expensive equipment is
required Measures total organic nitrogen not
just protein N