Proteins Flashcards
Why test for proteins?
Hypoproteinaemia: kidney disease (proteinuria), liver disease, malnutrition
Hyperproteinaemia: Multiple myeloma, dehydration
Monomer of proteins?
Amino acids
What are large organic molecules made up of monomers called?
Macromolecules
What are amino acids made up of?
Central carbon atom (alpha carbon) attached to:
>Hydrogen atom
>Amino acid (NH)
>Carboxylic acid group - COOH
>R group (20)
How do amino acids bond to form protein?
Via peptide bonds - the COOH group of one AA covalently bongs to the NH of another AA via a condensation dehydration reaction
What determines the protein’s function?
It’s final shape (tertiary or quaternary structure)
Name some diseases caused by misfolded proteins
Cystic Fibrosis, Alzheimer’s, Creutzfeldt-Jajob disease
What type of disease and what cause BSE?
Transmissible spongiform encephalopathy - caused by prions which cause other prions to misfold
What is a chaperone protein?
Ensure proteins fold correctly
How many stages of protein structure are there?
4 (some only have 3)
What is a prion?
An infectious misfolded protein which causes other proteins of the same kind to misfold
What is the composition of a protein?
Made up of carbon, hydrogen, oxygen, nitrogen (12-18%) & ~1% sulphur
>Molecular mass of 10,000-1 million g/mol
What percentage of albumin is made up of nitrogen?
16%
Cellular role of protein?
Transport & storage of other molecules, structural framework, antibodies & blood clotting factors, enzymes
What role do proteins play in support?
Collagen in ligaments, tendons & skin
Keratin in hair & nails
What are enzymes
Majority are proteins. Catalyst speed up chemical reactions.
What role do proteins play in transport?
Haemoglobin in blood, membrane proteins
What role do proteins play in immunity?
Antibodies are proteins
What role do proteins play in regulation?
Insulin - regulates blood sugar
What role do proteins play in motion?
Contractile proteins e.g. actin & myosin cause muscle movement
Reference range in recumbent patients
62-82 g/L
Proteins make up ~7% of the mass of serum (91% solvent & 2% nutrients)
What method is used generally to measure total protein level?
Colorimetry
Explain the Kjeldahl Titration method
> Quantitative determination of organic nitrogen in chemical substances e.g. ammonia
Nitrogen from protein & other nitrogen containing components are converted to ammonium ions (NH4+) by treating sample with sulphuric acid, potassium sulphate, & copper sulphate
Alkalisation with NaOH liberates ammonia vapours - distilled into boric acid solution - titrated with a standard acid to determine percentage nitrogen
Explain the Biuret method
> Biuret method - strong solution of NaOH & KOH with 1% copper sulphate
Presence of peptides - copper (II) ions form violet complexes in alkaline solutions
1 copper(II) ions: 6 peptide bonds
Deeper purple = more peptide bonds (more proteins) - spectrophotometry
Explain the Phenol Method
> Phenolic side chain of tyrosine & tryptophan in protein reduce phosphomolybdotungstic acid to form a blue colour
More sensitive than Biuret - inaccurate in specimens with abnormal protein content
What is the Folin-Lowry method?
A mix of Biuret and Phenol method - 100x more sensitive than Biuret alone
Explain the Turbidimetric & Nephelometric Method?
> Precipitation of protein with sulphosalicylic acid(SA)/Trichloroacetic acid(TCA)/Both together
Fine precipitates of insoluble protein - scatter incident light in suspension
Negative charge SA - neutralize positive charge in protein - denaturation & precipitation
Explain Refractometry
> Refractive index of sample measured relative to refractive index of water
Inaccurate in protein concentrations >35g/L
Must be diluted for <110g/L with equal parts water
Determine total serum proteins before Serum Protein Electrophoresis
Determination of Total Protein in Urine
> Biuret method - acid precipitated proteins/concentrate from membrane filtration - equally sensitive to each protein
Turbidimetric & dye binding methods - less time consuming
Reference range of spot urine
<140mg/24 hours
Determination of Total Protein in Cerebrospinal Fluid
> Turbidimetric methods & Coomassie Brilliant Blue dye-binding method (Bradford method)
Turbidimetric method - requires 0.2-0.5ml
CBB - as small as 25 ul
CSF Proteins reference range
200-400 mg/L
Elevated Proteins in CSF
Infectious, intracranial haemorrhages, multiple sclerosis, Guillian Barré syndrome, malignancies, endocrine abnormalities, certain medication, some inflammatory conditions
Reduced proteins in CSF
Repeated lumbar punctures or chronic leak of CSF
Components of an electrophoresis system
1) Support media
2) Power supply
3) Electrodes and chambers
4) Dryer
5) Visualisation
6) Densitometer
Albumin Method
> Dye-binding method (Bromocresol green) - certain chromogenic dyes form complexes with albumin
Dye must 1) absorbance when bound to albumin must be a different wavelength than unbound dyes 2) complex should absorb wavelength where haemoglobin & bilirubin have minimal interference 3) dye binds to albumin specifically, not other serum constituents
Methyl orange & Bromocresol purple can also be used
Albumin reference range
35-52 g/L
Explain Electrophoresis
> Movement of ions under the influence of an electric field
Cations and anions in aqueous solution travel towards the opposite charge - cations(+) to cathode(-), anions(-) to anode(+)
Large molecules (proteins) separated by electrophoresis
How are proteins and amino acids amphoteric?
Act either as proton donors (acid) & proton acceptors (base)
How do proteins act in a basic solution?
They lose protons to form negative ions. More acidic R-group residues = more negative charges on protein = more rapidly protein fractions migrate on a solid medium towards a positive pole of an electric circuit
In protein electrophoresis, explain the support media
Usually cellulose acetate or agarose gel-coated plastic plates
In protein electrophoresis, explain the power supply
> Provide a constant voltage (50 - 200V)
Higher voltage - improve resolution & decrease time taken - can heat up buffer & melt gel
In protein electrophoresis, explain the electrodes and chambers
> Combined into a single unit
Anode & cathode lie into divided buffer chamber - electrodes are made of graphite or metal
Divided chamber prevents completion of an electrical circuit until submerged fully in buffer
Covers over the buffer chamber - minimise evaporation
In protein electrophoresis, explain visualisation
> Some macromolecules - haemoglobin intensely coloured
Staining can be done on native plates (lipids,proteins)/ deferred until chemical or enzymatic reactions produce a stainable compound
Some fluorescent - visualised under UV
Visual inspection identify & crudely quantify bands on a plate
In protein electrophoresis, explain the densitometer
> Spectrophotometer - measure light transmittance through a solid sample
The plate is positioned over the monochromator - can scan plates - plates moved across the exit slit
What is the general procedure for electrophoresis?
> Cellulose acetate medium is soaked in a barbitol buffer (pH 8.6)
After blotting - samples & control applied
Cellulose acetate strip placed in Electrophoretic chamber - buffer added - current applied
After - stained with Ponceau S (red)
Quantitation - scanned on densitometer
Order of bands in electrophoresis strip
1) Albumin
2) Alpha-1
3) Alpha-2
4) Beta
5) Gamma
A note on albumin
> 50-60% total plasma proteins - main protein produced by liver
Maintain osmotic pressure
Carrier via reversible binding to bilirubin, lipids, hormones, metals, drugs
A note on Alpha-1 globulins
> Main component alpha-1-antitrypsin (produced by liver - protects lungs)
Deficiency - liver disease & childhood pulmonary emphysema
Also include Glycoprotein and alpha-1-lipoprotein (HDL)
A note on alpha-2-globulins
> main component alpha-2-macroglobulin (inhibit protease & act as carrier)
Increased in nephrotic syndrome
Include haptoglobulin & caeruloplasmin
A note on beta-globulins
VLDL, transferrin, complement
A note on Gamma globulins
Immunoglobulins
Multiple myeloma in protein electrophoresis
An extra M band between beta and gamma globulins
