Lecture 8- Protein Analysis Flashcards
What is protein analysis important?
Nutrition
Funtional properties
Economic consideration
What are the subunits of proteins?
Amino acids
What are the different structures of proteins?
Primary, secondary, tertiary, quaternary
What polypeptide bond links amino acids?
Peptide bond
What are the Non polar amino acids?
Glycine Alanine Valine Leucine Methionine Isoleucine
What are the polar amino acids?
Serine Threonine Cysteine Proline Asparagine Glutamine
What are the Aromatic amino acids?
Phenylalanine
Tyrosine
Tryptophan
What are the positively charged amino acids>
Lysine
Arginine
Histidine
What are teh negatively charged amino acids?
Aspartate
Glutamate
What is the distinguishing element in proteins?
Nitrogen
What is the range of nitrogen content in food proteins?
13-19%
What are the basic principles for protein determination?
- determination of N
- Peptide Bonds
- ARomatic Amino acids
- Dye binding capacity
- Ultraviolet absorptivity
What is the %Crude Protein?
Total N x Conversion Factor (~6.25)
What is total N?
Non Protein Nitrogen (NPN)
+
True Protein Nitrogen (TPN)
What are some examples of Non protein nitrogen?
Free amino acids Peptides Phospholipids Amino sugar nucleic acids Urea Nitrates Nitrites
In proximate analysis, what proteins content do we measure?
Crude , not True
What is the converstion factor?
Nitrogen to protein conversion assumes that 1kg of plant or animal protein contain a specific amount of nitrogen
6.25
If the nitrogen content ranges from ____% that means that nitrogen content is ___g/kg
13-19%
130-190g
What is the conversion factor for eggs? Wheat? Soy?
egg-6.25
wheat-5.33
soy-5.52
Calculation the converison factor for eggs
16% N therefore 1Kg egg proteins contains 160g N
1000/160=6.25
What are the two methods to determine crude protein?
Kjeldahl method--(N measured as ammonia NH3) Dumas Method (N measured as N2)
What is the Kjeldahl method based on?
Conversion of organic nitrogen (Total N) in food sample to ammonia (NH3)
What are the two steps in the Kjeldahl method?
Digestion: converts all N to NH3
Measurement of NH3: 1) distillation followed by titration 2) Colormetic methods 3) ammonium electrode technique
What happens in the digestion step?
Sample(N) +Conc H2SO4 (@300-400C)—–> CO2+ NH4+SO4+H2O
(release some toxic gas)
NH4+H2SO4–> H2SO4
( Clear solution, very stable salt in acid solution, nitrogen trapped in ammonium salts)
What is the first part of digestion?
PRotein nitrogen is liberated to form NH4 ions
H2SO4 oxidizes organic matter and combines with NH4 to form non volatile ammonium sulfate NH4SO4
Carbon and hydrogen elements are converted to CO2 and H2O
What does the catalyst do in Kjeldahl digestion?
Speeds up digestion/oxdiation
Seres and O carrier in oxidation
What are the catalysts used in Kjeldahl digestion? (3)
- Mercury oxide–> toxic, no longer used
- Selenium
- Copper
Why is K2SO4 added to Kjeldahl digestion?
The boiling point of H2SO4 is 290C, not enough to convert all protein N ot NH3 –> INCREASE BP
How does Kjeldahl method measure NH3 via distillation? (equation)
(NH4)2SO4+ 2NaOH or (KOH)–>2NH4OH+ Na2SO4
2 NH4OH–> 2NH3 + H2O–> (added to the first equation)
then…
NH3+ H3BO3–>NH4 + H2BO3
How does Kjeldahl method measure NH3 via distillation? (words)
Addition of NaOH or KOH increases pH of solution to above 9.2
Some NH4 forms complexes with metals and Hg NH5 conplexes.
Why is Sodium Thiosulfate added to the distillation technique of NH3 determination in kjeldahl method?
Addition of Sodium thiosulfate breaks these complexes asfollows:
NH2-Hg +Na2S2O3–>(H+)
NH3+HgS2O3
What is the titration equation in NH3 determination of the Kjeldahl method?
Borate anion is titrated with diluted HCl
H2BO3 + H–>
H3BO3
How do you calculate the % protein from the titration?
Moles of HCl= moles of NH3 = moles of N in the sample %N = N HCl x Corrected acid volume x 14/ g of sample x 1000 X 100 %N x 6.25 = % protein
What is the Colorimtirc method of NH3 determination?
NH4 can form colored compounds with different reagents
What are the two reactions with the coloremetric method?
Nessler Reaction
Berthelot Reaction
What is the Nessler Reaction? + colour
4NH4OH + 2HgI2+ 4KI + 3KOH —>
NH2Hg2IO + 7KI + 2H2O
Ammonium dimercuric iodide, red-orange, 440nm
What is the berthelot reaction? + colour
NH3+ phenol + hypochloride
Indophenol (Blue, 630nm)
What is the Ammonium ion electrode?
Ammonium ions are measured by ion selective electrode (ISE)
–> depends on ionic strength
Is ISE specific to an ion?
yes and no
Designed to respond to specific ion but often other ion will interfere
What are the disadvantages of the ammonium ion electrode?
- no used frequently
- low reliability
- expensive
What are the advantages of the Kjeldahl method?
Widely used and internationally accepted (AOAC)
Used as standard method for comparison against all other methods
High precision and good reproducibility
What are the disadvatnages of the kjeldahl method?
Is not a measurement of the true protein content (low accuracy)
Different proteins need different convertionfactors because they have different amino acid sequences.
Use of hazardous reagents
Time consuming to carry-out.
What is the Dumas method?
Combstion method, based on the conversion of organic nitrogen in the food sample to N2
What are the two steps in the Dumas method?
- combustion
2. Measurement of elemental N2
What are the reactions in the the Dumas method?
Sample N –> (875-900, oxygen gas) –> NO, NO2, N2O + H2O+SO2+N2+ CO2 —> (reductin of NO by Cu) –> H2O +SO2+N2+CO2–> N2–> Measurement of % Total N
What are the steps in the Dumas method?
Samples (approximately 100–500 mg) are weighed into a tin capsule
Introduced to a combustion reactor
Combustion under high temperature (9000C) in presence of catalyst (CuO/V2O5)
Volatile decomposition products (mainly molecular nitrogen, nitrogen oxides, carbon
dioxide, and water vapour) are transported by the carrier gas
Nitrogen oxides are reduced to molecular nitrogen and the excess of oxygen is bound to the copper or tungsten in the reduction column at a high temperature 6000C
Water is removed by means of a condenser filled with magnesium perchlorate,
diphosphoruspentoxide or other drying agents
Interfering compounds (e.g. volatile halogen and sulphur compounds) are removed by
absorbents materials (e.g. silver wool)
The nitrogen in the residual gas mixture, consisting of nitrogen and carrier gas, is passed through a thermal conductivity detector.
What adulterants are used to increase N content in food?
Urea, ammonia, melamine
Do the Kjeldahl and Dumas method detect adulteration?
No
What are the steps of Trichloro acetic acid (TCA)
- Precipitate out proteins from a solution or dry sample using 10% TCA
- Mix the reaction mixture thoroughly and let precipitate settle for 5 min.
- Filter the mixture through WhatmanNo. 1 filter paper or centrifuge at 30,000g to retain the proteins while other components are washed away.
- Determine nitrogen content of the filtrate by the Kjeldahlmethod.
- Convert nonproteinnitrogen to protein equivalent with conversion factor.
What is the Biuret Method?
Depends on the formation of violet to purple color when Cu2+ ioins from a complex with peptide bonds under alkaline conditions
What is mixed in the Biuret reagent?
How is the sample analyzed?
(CuSO4+ NaOH + potassium sodium tartrate) and allowing to stand for 15-30 min, then the absorbance is measured at 540 nm using spectro-photometer
Protein Sample + Biuret reagent –? Chelate Complex
What are the Biuret Reagents+ their roles?
- NaOH or KOH - alkaline conditions
- Copper sulfate CuSO4 provides Cu2+ ions
- Potassium sodum tartrate stabilizes the complex
What is a positive test of the Biuret
blue –> purple (proteins)
Blue –> pink (peptides)
What is a negative biuret test?
No change in colour (stays blue)
What is this the Biuret sensitive to? What is measured?
Biuret is selective to peptide bonds, not sensitive
Only measures proteins and peptides
What is the Lowry method?
Combines Biuret and Folin-Cicocalteau pehnol reagent (phosphomolybdic and phosphotungtic acid)
What is the principle of the lowry method?
Cu+ produced after reduction of Cu2+ by peptide bonds in complexed with folin-cicocalteau phenol regent to form a stable blueish color which is measured at 500 or 750 nm
What is reduced in the lowry method?
Phosphomolybdotungstateis reduced to heteropolymolybdenum
What is the Lowry method sensitive to?
Reaction is pH sensitive…pH should be 10-10.5
What is the Bicinchonic Acid Method? (BCA)
Proteins reduce cupric ions (Cu+2) into cuprous ions (Cu+1) under alkaline conditions; then the Cu+1 reacts with BCA reagent to form a purple complex which is measured colorimeticallyat 562nm.
Protein + Cu2–> Cu1
What contributes to the colour in the BCA method?
Peptide bonds and 4 amino acids (Cysteine, cystine, tryptophan, and tyrosine)
Show the steps of the BCA method
insert photos, slide 36
BCA sodium salt -> BCA copper reaction
What is the Anionic Dye Binding Method?
Protein containing sample is mixed with a known excess of a negatively charged anionic dye ina buffered solution
The proteins form an insoluble complex with the dye because of the electrostatic attraction and the unbound (free) dye remains soluble.
The remaining amount of the unbound dye is separated (e.g. by centrifugation) and then determined by measuring its absorbance.
Explain with a flow chart the Anionic Dye binding method
Protein+ excess dye –> Protein-dye complex (insoluble)+unbound solube dye–> filtrate or the supernatant contain the unbound soluble dye–. unbound soluble dye is measure
How do you determine the bound dye?
Bound dye = excessdye-unbound dye
What is the Bradford Method?
CoomassieBrilliant blue G-250 binds electrostatically to proteins; this leads to changing the dye’s color from reddishto bluish.
The change in the absorbance at 595nm is proportional to the protein concentration of the sample.
What is the Ultraviolet 280nm Absorption method
Protein solubilized in buffere or alkali show strong absorption to region of ultreaviolet radiation at 280nm, due to tryptopha nand tyrosine residues in the proteins.
What is the flow chart of the ultraviolet 280nm absorption method?
UV source–> UV is absorbed by proteins at 280nm–> protein contain aromatic amino acids
What is a disadvantage to the 280nm absorption method?
The major disadvantage of this method is that nucleic acids also absorb UV strongly at 280 nm and could therefore interfere with the measurement of the protein.
Which is more sensitive - the lowry or biuret method?
lowry
