What is protein:
• polymer of 20 a– amino acids
• N is most distinguished element: among the composing elements of C,H, N, O, S, for some proteins: P, Cu, Fe, I.
• N content in different proteins ringing from 13.4% -19.1%, and averagely 16%.
Ó Proteins are important components of food, providing biological and structural function.
Ó There importance for analysis is primarily related to their unique biological activities or functional properties, and for nutritional benefits.
Protein analysis: You need to know..
1. total protein
2. amino acid composition
3. amount of a particular protein in a mixture
4. protein content during isolation and purification
5. Nonprotein nitrogen
6. Nutritional value of a protein
Soluble & Insoluble Protein – Kjeldahl & Dumas (combustion) – measures total nitrogen (N)
Soluble protein – Biuret, Lowry, UV, Dye binding, Bradford, Ninhydrin
• Based on conversion of protein nitrogen to ammonia (NH3). At the same time, carbon and hydrogen are oxidized to carbon dioxide and water.
• In the presence of sulfuric acid (H2SO4), the ammonia picks up a hydrogen forming ammonium sulfate (NH4)2SO4.
• Then concentrated sodium hydroxide (NaOH) is added to the solution of ammonium sulfate + sulfuric acid. This raises the pH transforming ammonium (NH4+) into ammonia.
• When the sample containing ammonia, sodium hydroxide and sodium sulfate is heated, the ammonia is driven off as a gas.
• We can condense the ammonia gas and introduce it into a fluid that contains boric acid
• Ammonia reacts with boric acid to form ammonium borate. This is a base.
• Titrate ammonium borate to an endpoint with hydrochloric acid (we must know the normality of the hydrochloric acid used).
3 Stage Process:
• 1. Digestion with acid + catalysis
• 2. Distillation with steam and alkali
• 3. Titration with acid and indicators.
l Protein + H2SO4 à (NH4)2SO4 (Digestion)
l NH4+ + NaOH àsteam and heatà NH3 (g) + H2O (Distillation)
l NH3 (l) + H3BO3 à NH4+ + H2BO3- (Trapped)
l H2BO3- + HCl à H3BO3 (Titration)
KJELDAHL Advantages are
1. applicable to most food samples
4. accepted as Official method
5. can measure mg levels of proteins
1.Measures total N not protein
2.Time consuming – at least 2 hrs
3. Poor precision when compared to other methods
4. Corrosive (dangerous) method
• Applications: Suitable for cereals, meat, soybean, and isolated proteins
– Cheaper and faster than Kjeldahl
– Less problem with color deviations than other protein methods
– Few substances interfere
– Does not measure non-protein nitrogen (NPN)
– not sensitive, only to 2-4 mg level
– bile pigments interfere
– ammonium salts interfere
– color depends on type of protein
– lipids and carbos can affect clarity of solution
– PROTEIN MUST BE SOLUBLE
• Going to test soluble protein against a standard of BSA.
• The reaction of peptide bonds in protein with copper ions in alkaline conditions will produce a purple color.
• The intensity of the color is directly proportional to the amount of protein.
• Quantify using a spectrophotometer (540 nm)
The lowry’s Method (Folin-phenol method)
Folin reagent phosphomolybdic and phosphotungstic acid is reduced to a blue molybdenum complex, mainly by the phenolic groups of tryptophan and tyrosine.
Lowry greatly increased the sensitivity of the determination by preceding the reaction by pretreatment with a copper reagent in a basic medium.
DYE BINDING METHOD
l Proteins will bind to certain types of dye. When this binding occurs, the protein-dye complex will precipitate.
l The unbound dye is then easily determined with a spectrophotometer using a standard curve with varying dye concentrations.
l Using the amount of dye initially added to the protein solution, the amount of protein can be calculated.
Bradford assay-Coomassie Brilliant Blue
• 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.
• Rapid assay
• Useful when accuracy is not crucial
Applications: Finds use as rapid protein method in food and biomedical industry.
• 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.
• Faster and more convenient that Kjeldahl
• 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.