Title: Protein Analysis
1Protein Analysis
Chapter 9
2Other Common Protein Methods
3Biuret 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
4Biuret 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
6LOWRY 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.
7LOWRY 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.
8LOWRY 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
9LOWRY 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
10UV 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.
11Amino Acid Structures
PHENYLALANINE
TYROSINE
TRYPTOPHAN
12UV 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?
13UV 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
14UV 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
15UV 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.
17Bradford protein assay - showing increasing
amounts of protein concentration
18Ninhydrin
- 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
21Dumas 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.
22Dumas 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