Analysis of Proteins Flashcards
Importance of analysis
Enzymes
Enzyme inhibitors
Functional proteins
Nutritional labeling
Kjeldahl
Digested with sulfuric acid + catalyst
Organic N –> ammonium sulfate
Neutralized with base
Distilled into boric acid
Create borate anions
Titrated with acid
Crude protein content
Kjeldahl Sample Prep
Solid foods are ground
Samples should be homogenous
No other prep
Kheldahl digestion
Nitrogen –> ammonium ions
C and H elements –> CO2 + water
Kjeldahl neutralization + distillation
Diluted with water
Sodium thiosulfate neutralizes
Ammonia distilled into acid
Kjeldahl calculation
Mol HCl = mol NH3 = mol N
Kjeldahl Advantage
All types of food
Simple
inexpensive
Accurate
Official method
Small quantities of sample
Kjeldahl disadvantages
Total organic N (not protein N)
Time-consuming
Poorer precision than biuret
Corrosive reagents
False + results
Bias from adulterants
Biuret Method
Violet purple color
Copper ions complex w/ peptide bonds
Read absorbance
BSA curve
Cereal, meat, soybean `
Biuret advantages
Less expensive than Kjeldahl
Rapid
Simple
Very few interferences
Does not detect non protein N
Biuret Disadvantages
Not very sensitive
Color varies w/ diff proteins
High concentration of ammonium salts interfere
Opalescence (high fat/carb)
Lowry method
Biuret + folin ciocalteau
Tyrosine and tryptophan residues
Read blue color
BSA curve
Lowry advantages
Sensitive
Less affected by turbidity
Disadvantages
Color varies w/ different proteins
Color is not proportional to [protein]
interference
BCA
Peptide bonds reduce Cu ions under high pH
Color change from green to purple
Phospholipids interfere
UV
Tryptophan and tyrosine residues show absorption
Need a pure system
Bradford method
Brilliant blue binds to protein
Color change (red to blue)
Change in absorbance proportional to concentration
Beer, potatoes
Bradford advantages
Rapid
Reproducible
Sensitive
No interference from cations, ammonium sulfate, polyphenols
Bradford disadvantages
Complex binds to cuvettes
Use glass or plastic cuvettes
Color varies w/ proteins
Anionic dye binding
Protein binds dye to form precipitate
Unbound dye is measured after removal of precipitate
Unbound dye inversely proportional to protein
Milk, wheat flour, soy product, meat
Anionic dye advantages
Rapid
Inexpensive
Relatively accurate
Lys in cereals
Anionic dye disadvantages
Not sensitive
Need calibration curve for a given food
Interferences and errors
Rapid protein analyzer
Simple
Fast
No hazard chemicals
Approved
Unbound dye inversely proportional to protein
Ninhydrin
Purple color formed
Hydrolysis of peptide bonds
Quantitate AA
Rapid
Ninhydrin Disadvantages
Color varies w/ AA composition
Standard calibration curve needed
Dumas method
Combust at high temp in the presence of O2
Traps to move certain compounds
Dumas advantages
Alt to Kjeldahl
No hazardous chemicals
Quick
Can do many samples at once
Dumas disadvantage
Expensive equipment
Nonprotein N included
IR
Mid or near IR
Expensive
Rapid
Grain, cereal, meat, dairy
Separate proteins by
Size
Charge
Absorption
Solubility
Heat stability
Solubility characteristics determined by
Type and charge of AA
Precipitate proteins by
Buffer
pH
Ionic strength
Dielectric constant
Temp
Salting out
Proteins precipitate out as ionic strength is increased
Isoelectric precipitation
Proteins aggregate at pI
Solvent precipitation
Separate by solubility in different solvent mixtures
Acetone and ethanol decrease solubility (mostly)
Separation by adsorption
Ion exchange chromatography
Affinity chromatography
Ion exchange chromatography
Column has a certain charge
Proteins with that same charge exit first
Proteins with the opposite charge stick to the column and come out last
Affinity chromatography
Column has a ligand for a certain protein
Unwanted proteins are washed through
Protein sticks to the ligand
Release with elusion
Dialysis
Use semipermeable membranes
Low MW solutes diffuse out
Ultrafiltration
Similar to dialysis
Semipermeable membranes with cutoffs
Molecules larger than the cutoff are retained
Molecules smaller than the cutoff pass through the membrane
Size exclusion chromatography
Agarose or dextran = stationary phase
Molecules larger than the pores are excluded, leave 1st
Small molecules enter the pores and are retained
Electrophoresis
Migration of charged molecules in a solution through an electric field
Polyacrylamide gels
Small proteins migrate faster
Capillary Electrophoresis
Uses really thin capillary tubes
High efficiency separation
High electric field strengths
Minute amounts of sample
Precise quantitative analysis
Amino Acid Analysis
Directly measure or estimate the essential AA content
Measure how well a protein is Digested, Absorbed, Utilized
Protein Efficiency Ratio
Based on weight gain of rats
Test protein vs control diet
How much protein is used to build muscle, absorbed
Net Protein Ratio
Value of a test protein for cell maintenance
Biological Value
Absorbed N retained for maintenance or growth
Corrected for metabolic losses
True digestibility
Based on amount of N ingested and feed intake
Corrected for losses in the feces
PDCASS
AA score x true digestibility
Solubility test
Find concentration where the protein is no longer soluble
Emulsification
Tested by centrifugation or agitation
Wait for separation
Foaming
Volume and stability are important
Overrun measures the air incorporated into the foam
Gelation and gel formation
Measure rheological properties
Mixograph and farinograph
Nitrogen titration
%N =
(Volume - Blank)(Normality)(MW) / Sample Mass
(All x 100%)
