Analysis of Proteins Flashcards by PAYTON GLADEM

Importance of analysis

Enzymes
Enzyme inhibitors
Functional proteins
Nutritional labeling

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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

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Kjeldahl Sample Prep

Solid foods are ground
Samples should be homogenous
No other prep

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Kheldahl digestion

Nitrogen –> ammonium ions
C and H elements –> CO2 + water

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Kjeldahl neutralization + distillation

Diluted with water
Sodium thiosulfate neutralizes
Ammonia distilled into acid

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Kjeldahl calculation

Mol HCl = mol NH3 = mol N

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Kjeldahl Advantage

All types of food
Simple
inexpensive
Accurate
Official method
Small quantities of sample

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Kjeldahl disadvantages

Total organic N (not protein N)
Time-consuming
Poorer precision than biuret
Corrosive reagents
False + results
Bias from adulterants

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Biuret Method

Violet purple color
Copper ions complex w/ peptide bonds
Read absorbance
BSA curve

Cereal, meat, soybean `

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Biuret advantages

Less expensive than Kjeldahl
Rapid
Simple
Very few interferences
Does not detect non protein N

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Biuret Disadvantages

Not very sensitive
Color varies w/ diff proteins
High concentration of ammonium salts interfere

Opalescence (high fat/carb)

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Lowry method

Biuret + folin ciocalteau

Tyrosine and tryptophan residues
Read blue color
BSA curve

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Lowry advantages

Sensitive
Less affected by turbidity

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Disadvantages

Color varies w/ different proteins
Color is not proportional to [protein]
interference

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BCA

Peptide bonds reduce Cu ions under high pH

Color change from green to purple
Phospholipids interfere

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Tryptophan and tyrosine residues show absorption

Need a pure system

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Bradford method

Brilliant blue binds to protein
Color change (red to blue)
Change in absorbance proportional to concentration

Beer, potatoes

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Bradford advantages

Rapid
Reproducible
Sensitive
No interference from cations, ammonium sulfate, polyphenols

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Bradford disadvantages

Complex binds to cuvettes
Use glass or plastic cuvettes
Color varies w/ proteins

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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

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Anionic dye advantages

Rapid
Inexpensive
Relatively accurate
Lys in cereals

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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

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%)