(12) Protein Analysis Flashcards
What is a proteome?
The functions of proteins in a cell, particularly at the metabolic and regulatory level
- applies to any cell, whether of single cell organisms (bacteria) or of multi cellular organisms (plants and animals)
Applications of proteomics
- production and purification of natural enzymes for use in industry
- Genetic enginerring of industrially important cells for food, agriculture, medical and environmental application
- Improved analytical techniques
4, Human health: purified protein for medical treatment, improved pharmaceuticals for human and animal by better target selection and reduced side effects
list the enzymes (protein) used in the food industry
- carbohydrase (lactase, pectinase)
- protease
- lipase and esterase
- oxidoreductase
- lyase
- transferase
How is protein function determined?
By its primary structure, the sequence of amino acids in the polypeptide string
this occurs in part due to the sequence specifying how the polypeptide will fold to form the structure of the protein in 3D
General structure of amino acid
glutamic acid, lysine, arginine
- basic building blocks of proteins; consists of an alpha carbon, an amino group, a carboxyl group and a R-group attached to the center C R(NH2)CO2H
- optically active and can be L or R (L are biologically active)
- AAs(like proteins) are amphoteric (acts as acids and alkaline)
- the type of amino acids which the protein is composed determines the functionality of that protein
Discuss peptide bonds
- The amino group of one amino acid reacts readily with the carboxyl group of another amino acid
results in one molecule of water being eliminated to have 2 amino acids joined (peptide bond) - Polypeptides: longer chains of amino acids
Protein quantification techniques
- Kjeldahl method
- Dye binding method
- Biuret method
- Lowry method
- Ultraviolet method
- Fluorescent method
Discuss the Kjeldahl method + mechanism
main method to quantitate all protein in a given sample (doesnt have to be food)
- break down of sample (release nitrogen) with concentrated H2SO4 and a catalyst
- the nitrogen in the sample is converted into an ammonium ion using boric acid
- The solution is neutralized with NaOH to get NH3
- Steam distillation of NH3 and trap boric acid
- the solution is titrated with HCl to calculate the amount of N2
Disadvantages of Kjeldahl method
In cells, not all nitrogen is in protein
> Nitrogen can be come from purine, pyrimidine or urea
> many plant tissues have more than 50% non-protein Nitrogen
!!!!!! Hence, a conversion factor for protein in food is used to determine protein content,
Discuss the dye binding method
Principle:
at low pH, basic groups of protein are positively charged; these will quantitatively bind a negatively charged dye
Procedure
- Mix protein, dye and pH 2 buffer
- Filter or centrifuge
- Measure optical density of filtrate (470nm)
factors affecting quantification
- temperature
- protein quality (including non-protein)
- Buffer systems (pH, salt concentration)
Discuss the biuret method
Under alkaline conditions, substances containing two or more peptide bonds form a purple complex with Cu++ (copper salts) in Biuret reagent
- the colour intensity is proportional to the protein content of the sample
- the intensity of the colour is measured at 540nm
Discuss the modified lowry method
Principle:
combines the biuret reaction with the reduction of the folin-ciocalteau phenol reagent by tyrosine and tryptophan residues in the proteins > deep blue colour is measured at 750 nm
Discuss the UV absorbance method
- Detection of chromophoric side chains of aromatic acids includes phenylalanine, tyrosine, tryptophan
- Absorbance is measured at 280nm (non-destructive way to determine protein)
- Calculation of protein concentration is based on absorption value
DISADV:
concentrations of aromatic amino acids are generally low in most proteins
Discuss the fluorescence method
hit sample with special wavelength and measure amount emitted
- Tyrosine and tryptophan are fluorescent compounds
- amino acids are excited at 280nm
- the emission is measured at 348nm
Advantage: a more sensitive technique than UV absorption
Principles of protein separation
A particular protein may be purified from a cell extract or other protein mix based on character differences between proteins such as
mass (MW), charge (isoelectric point), hydrophobicity, affinity to substrate-like compounds or others
Techniques used to separate protein by mass
Gel: SDS 1D polyacrylamide gel electrophoresis
Column: LC or HPLC
Discuss SDS 1D Electrophoresis
Sodium dodecyl sulphate (SDS) an anionic detergent is used to hydrolyse proteins and give them similar charges per molecular weight (protein will unfold and carry negative charge)
- proteins in acrylamide gel separate under an electrical field, according to molecular weight
- under an applied electrical field and due to net negative charges they migrate form the cathode (-) to the anode (+)
- during electrophoresis, proteins form separate bands in the acrylamide gel according to their molecular weights (small MW migrate faster and reach high Rf)
General stains are used to expose protein bands in the gel
- Coomassie blue (µg sensitive)
- silver ions (ng sensitive)
Limitations of 1D electrophoresis
- A maximum of 30 proteins can be separated (identified) in 1 lane
- not all proteins bind to SDS well (membrane proteins), these proteins may form smears on a gel rather than tight bands or may not become soluble (wont enter gel)
Advantages and disadvantages of biuret and lowry compared to micro-kjeldahl
In the biuret reaction, the cupric ion complexes with peptide bonds of polypeptides and proteins to form a change in colour in alkaline solution, the intensity of colour change is directly proportional to the concentration of protein, this colour change is rapid and does not fad quickly, this however only reacts with peptide bonds and not the side chains, hence it can only detect soluble proteins
For the lowry method, it can only detect soluble protein as well and the colour intensity is proportional to the protein concentration, and it is more sensitive than the biuret method where tyrosine and tryptophan mole fractions will give a higher colour intensity, since it is more sensitive to these two amino acid, a proper standard curve have to be plot to avoid inappropriate quantification of protein content
the lowry method is also prone to interference by contaminating acids leading to a false positive
the colour formed is relatively slow and fades quickly
The kjedahl method quantifies nitrogen content including those that are from non-protein sources such as urea
protein structure
- Primary: order of amino acids in the protein molecule
- secondary: further linking of amino acids by various bonds gives the protein a definite shape (often spiral)
- tertiary: folding of amino acid structure onto themselves, form a globule; usually there are hydrophilic residues on the surface and hydrophobic on the interior
- globular proteins often coiled further and appear in nature in the fibrous form
- made up of more than one tertiary unit
- insoluble in water, not greatly affected by alkalis, acid and heat
- keratin, elastine
difference between biuret and lowry method
both involves interaction between copper and amino group in alkaline conditions BUT lowry produces another reagent which interacts with aromatic AA (more sensitive)
