1. Chapter 7: Analysis of specific proteins & protein quality

2. Proteins are polymers of some 21 different amino acids joined together by peptide bonds. Because of the variety of side chains that occur when these amino acids are linked together, the different proteins may have different chemical properties and widely different secondary and tertiary structures

3. The side chains may be polar or nonpolar
The side chains may be polar or nonpolar. High levels of polar amino acid residues in a protein increase water solubility. The most polar side chains are those of the basic and acidic amino acids. These amino acids are present at high levels in the soluble albumins and globulins. In contrast, the wheat proteins, gliadin and glutenin, have low levels of polar side chains and are quite insoluble in water.

5. PROTEIN CLASSIFICATION
Simple Proteins
Simple proteins yield only amino acids on
hydrolysis and include the following classes:
• Albumins. Soluble in neutral, salt-free water. Usually these are proteins of relatively low molecular weight. Examples are egg albumin, lactalbumin, and serum albumin in the whey proteins of milk, leucosin of cereals, and legumelin in legume seeds.

6. Globulins. Soluble in neutral salt solutions and almost insoluble in water. Examples are serum globulins and (3-lactoglobulin in milk, myosin and actin in meat, and glycinin in soybeans.
• Glutelins. Soluble in very dilute acid or base and insoluble in neutral solvents. These proteins occur in cereals, such as glutenin in wheat and oryzenin in rice.

7. Prolamins. Soluble in 50 to 90 percent ethanol and insoluble in water
Prolamins. Soluble in 50 to 90 percent ethanol and insoluble in water. These proteins have large amounts of proline and glutamic acid and occur in cereals. Examples are zein in corn, gliadin in wheat, and hordein in barley.
• Scleroproteins. Insoluble in water and neutral solvents and resistant to enzymic hydrolysis. These are fibrous proteins serving structural and binding purposes. Collagen of muscle tissue is included in this group, as is gelatin, which is derived from it. Other examples include elastin, a component of tendons, and keratin, component of hair and hoofs.
• Histories. Basic proteins, as defined by their high content of lysine and arginine. Soluble in water and precipitated by
ammonia. .

8. Conjugated Proteins
Conjugated proteins contain an amino acid part combined with a nonprotein material such as a lipid, nucleic acid, or carbohydrate. Some of the major conjugated proteins are as follows:

9. • Phosphoproteins. An important group that includes many major food proteins, Phosphate groups are linked to the hydroxyl groups of serine and threonine,This group includes casein of milk and the phosphoproteins of egg yolk. • Lipoproteins. These are combinations of lipids with protein and have excellent emulsifying capacity. Lipoproteins occur in milk and egg yolk.

10. • Nucleoproteins. These are combinations of nucleic acids with protein
• Nucleoproteins. These are combinations of nucleic acids with protein. These compounds are found in cell nuclei. • Glycoproteins. These are combinations of carbohydrates with protein. Usually the amount of carbohydrate is small, but some glycoproteins have carbohydrate contents of 8 to 20 percent. An example of such a mucoprotein is ovomucin of egg white. • Chromopmteins. These are proteins with a colored prosthetic group. There are many compounds of this type, including hemoglobin and myoglobin, chlorophyll, and flavoproteins.

11. Derived Proteins
These are compounds obtained by chemical or enzymatic methods and are divided into primary and secondary derivatives, depending on the extent of change that has taken place. Primary derivatives are slightly modified and are insoluble in water; rennet coagulated casein is an example of a primary derivative. Secondary derivatives are more extensively changed and include proteoses, peptones, and peptides. The difference between these breakdown products is in size and solubility

12. These breakdown products are formed during the processing of many foods, for example, during ripening of cheese.

13. Protein structure

14. Analysis of specific proteins
Separation techniques
precipitation
adsorption
ion-exchange chromatography
affinity chromatography
size
dialysis
ultrafiltration
size-exclusion chromatography
electrophoresis
Non separation techniques
immunoassay
Nielsen 2003
Before separating proteins it is important to know about the biochemical properties of the protein, molecular weight, iso-electric point (pI) solubility properties, and denaturation temperature
To characterise and identify specific proteins of interest in terms of food quality:
species of origin - kangaroo meat being sold as beef
nutritional quality - vegetable protein sold as meat protein in processed food
allergic reactions - peanut proteins, gluten
functional/organoleptic properties - identifying genetic variants of dairy cows that produce milk with higher levels of specific foaming proteins for cappuccino milk!
Similar methods also used by food industry to prepare purified protein isolates as speciality food ingredients such as gelling agents in meat products and in pharmaceutical industry to purify biologically active proteins such as growth hormone for clinical use

15. Use of precipitation in protein analysis
Often the first step in protein separation
Separated proteins can then be more easily identified and characterised
Exploits differences in solubility of different types of proteins to partially purify protein of interest before identification
solubility determined by type and charge on amino acid -R groups
Nielsen 2003
Precipitation techniques (buffer pH, ionic strength, dielectric constant or temperature):
can deal with large amounts of material
relatively quick
other food components do not generally interfere
May be used to separate enzymes in meat that are linked to meat quality so they can be quantified individually

16. Proteins selectively precipitated by:
salting out (ionic strength)
use of neutral salts such as ammonium sulphate [(NH4)2SO4], NaCl and KCl
exploits lower but differing solubility's of different protein in increasing concentration of neutral salt solutions
iso-electric precipitation
iso-electric point, is pH at which protein has no net charge
exploiting different isoelectric points of proteins
Ionic strength - called ‘salting out’
Every type of protein has different level of solubility in neutral salt solution.
Ammonium sulphate, sodium chloride or potassium chloride is added to a mixed solution of proteins extracted from a food to a level just below that which precipitates the protein of interest
Solution centrifuged to remove types of proteins that have already precipitated.
More salts added to remaining solution to a concentration just above that which precipitates protein of interest.
Protein of interest removed by centrifugation leaving behind still soluble proteins in supernatant.
Disadvantage is that large quantities of salt contaminate the precipitated protein which should be removed through dialises
Buffer pH - separation based on fact that all proteins have different isoelectric point. Modify pH of solution of proteins to the isoelectric point of the protein in question. No net charge on protein so becomes insoluble and precipitates. Can then be separated by gravity settling, filtering or centrifugation from other proteins that remain soluble. Separated protein can be resolublised using buffer at different pH
Protein isoelectric point; Is the protein at which there is no overall charge on the protein, which results in minimum solubility
This is utilised in protein fractionation of protein solution (eg. Soya or lupin) without gross denaturation

17. Proteins selectively precipitated by:
solvent fractionation
protein solubility at fixed pH and ionic strength is a function of the dielectric constant
exploiting generally lower but differing solubility of most protein in water miscible organic solvents (ethanol, acetone)
Organic solvents (5% to 60%) decrease the ionization charge of amino acids
denaturation
exploits differential susceptibility of proteins to heat & extreme pH denaturation leading to precipitation
Solvent fractionation -
adding organic solvents such as ethanol or acetone decreases the dielectric constant of an aqueous solution and generally but differentially decreases solubility of most proteins by decreasing ionization of charged amino acids resulting in protein aggregation and precipitation of some proteins but not others at specific organic solvent concentration.
Solvent fractionation is preformed at 0°C to prevent protein denaturation caused by temperature increases that occur when organic solvents are mixed with water.
Denaturation of contaminating proteins -
Denaturation; proteins that are stable at high temperatures or at extreme pH are most easily separated by this technique, contaminating proteins are precipitated and proteins of interest remain in solution (more resistant to denaturation)
Soy protein may be precipitated from soy flour using 60-80% aqueous alcohol solution by iso electric precipitation at pH 4.5 or by denaturation with moist heat. Individually these methods produce concentrates of 65% two or three combined may produce concentrates above 90%

18. Separation of proteins by size
Proteins MW from 10, ,000,000 Dalton size
Actual separation depends on the stokes radius not on the molecular weight of the protein
Stokes radius: average radius of protein in solution
Dialysis
separation of proteins in a solution by selective diffusion through a semipermeable membrane
protein extract placed in sealed dialysis tubing in large volume of buffer (X ) for 12 hr.
small proteins leave bag larger proteins are retained
Nielsen 2003:
Stokes radius; For example a globular protein may have an actual radius very similar to its stokes radius, where as a fibrous or rod-shaped protein of the same molecular weight may have a stokes radius that is much larger than that of the globular protein. As a result the two proteins may separate as if they had different molecular weights
Dialysis- simple but slow method
Dialysis is used as part of protein separation sequence when trying to purify proteins

20. Separation of proteins by size
Micro, Ultra & Nanofiltration
differ in porosity & pressure
can separate proteins on basis of size using semi-permeable membrane under applied pressure
molecules larger than the membrane cut off are retained
Micro (0.1 to 5µm): remove particles & microorganisms
Ultra (0.005 to 0.1µm): concentrate and fractionate protein solution, remove salt
nano ( to 0.005µm): remove monovalent ions from salt whey
Micro (0.1 to 5µm): remove particles & microorganisms. Remove bacteria from milk and beer
Ultrafiltration is used as protein purification and concentration process and in commercial preparation of purified protein concentrates and isolates eg. whey protein concentrates that are high nutritional quality, highly soluble proteins from waste stream of cheese manufacture that are widely used in powdered drink mixes eg. Milo and in baked goods. A molecular weight cutoff membrane is used to remove lactose salt and water.

21. Use of electrophoresis in protein analysis
Separation depends on friction and charge of the protein.
Mobility = (applied voltage) (net charge on molecule)
friction of the molecule
proteins are positively or negatively charged
negatively charged when pH is above pI
positively charged when when pH is below pI
Magnitude of protein charge and applied voltage will determine how far a protein will migrate
Mobility decreases with increased frication due to higher stokes radius
The higher the voltage and stronger the charge on the protein the greater the migration within the electrical field. Molecular size and shape which determine the stokes radius of a protein, also determine migration distance within the gel matrix.
Smaller proteins migrate faster through gel matrix, a decrease in pore size of the gel matrix will decrease mobility
Polyacrylamide gel electrophoresis with an anionic detergent sodium dodecyl sulfate (gives negative charge to proteins therefore proteins are separated by size alone).
A reducing agent such as mercaptoethanol is used to reduce disulfide bonds within and between protein subunits

22. Used to determine protein composition of a food product
can identify how different food processing techniques can alter protein composition of soy protein isolates or whey protein isolates used as food ingredients
can identify species of meat used in foods
can determine purity of proteins eg enzymes used for food processing
individual proteins identified by their migration distance during electrophoresis
individual proteins quantified by the intensity of staining of bands on electrophoresis gel

23. The determination of the amino acid composition of proteins
Amino acid composition is used to:
assess nutritional quality of protein
characterise and identify a newly isolated protein
calculate nitrogen conversion factors for specific proteins
Food hydrolysed
Released amino acids separated chromatographically
Individual amino acids are identified and quantified
Amino acids separated using chromatographic methods Ion exchange, reverse phase liquid and gas liquid chromatograpny

24. Amino acid analysis - hydrolysis
Boiling 6N HCl for 24 hr to release amino acids
But some amino acids destroyed:
typtophan completely
methionine, cysteine, threonine and serine progressively
asparagine and glutamine converted to aspartic & glutamic acids respectively-not measured
isoleucine and valine linked peptide bonds hydrolysed more slowly
tyrosine may be oxidised
Hydrolyses all peptide bonds to constituent amino acids
Can heat on mantle with condenser or at smaller scale in sealed heat resistant glass bottles or ampoules
Typtophan - separately assayed chromatographically after alkaline hydrolysis
Methionine and cysteine can be measured by separate chromatogram after hydrolysis in performic acid to give cysteic acid then HCL hydrolysis and chromatography
Threonine and serine - zero-time levels estimated by sampling at three time (24, 48 and 72hr) intervals during hydrolysis, plotting level against time and interpolating line to zero time.
Asparagine and glutamine converted to aspartic acid and glutamic acid respectively - cannot be measured
Tyrosine may be oxidised - difficult to control
Expensive analysis - costs food company about $500 for full amino acid analysis of a sample by outside analytical lab. May need three separate hydrolysis chromatography runs to measure all amino acids accurately

25. Amino acid analysis – hydrolysis
Typtophan - separately assayed chromatographically after alkaline hydrolysis
Methionine and cysteine: separate chromatogram, hydrolysis in performic acid, give cysteic acid
HCL hydrolysis & chromatography
Threonine and serine – zero - time levels estimated by sampling at (24, 48 and 72hr) during hydrolysis
interpolating line to zero time.
Asparagine and glutamine converted to aspartic acid and glutamic acid respectively - cannot be measured
Tyrosine may be oxidised - difficult to control
Expensive analysis - costs food company about $500 for full amino acid analysis of a sample by outside analytical lab. May need three separate hydrolysis chromatography runs to measure all amino acids accurately