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1.1 Clinical Chemistry > Proteins > Flashcards

Proteins Flashcards

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1
Q

Why test for proteins?

A

Hypoproteinaemia: kidney disease (proteinuria), liver disease, malnutrition
Hyperproteinaemia: Multiple myeloma, dehydration

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2
Q

Monomer of proteins?

A

Amino acids

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3
Q

What are large organic molecules made up of monomers called?

A

Macromolecules

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4
Q

What are amino acids made up of?

A

Central carbon atom (alpha carbon) attached to:
>Hydrogen atom
>Amino acid (NH)
>Carboxylic acid group - COOH
>R group (20)

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5
Q

How do amino acids bond to form protein?

A

Via peptide bonds - the COOH group of one AA covalently bongs to the NH of another AA via a condensation dehydration reaction

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6
Q

What determines the protein’s function?

A

It’s final shape (tertiary or quaternary structure)

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7
Q

Name some diseases caused by misfolded proteins

A

Cystic Fibrosis, Alzheimer’s, Creutzfeldt-Jajob disease

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8
Q

What type of disease and what cause BSE?

A

Transmissible spongiform encephalopathy - caused by prions which cause other prions to misfold

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9
Q

What is a chaperone protein?

A

Ensure proteins fold correctly

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10
Q

How many stages of protein structure are there?

A

4 (some only have 3)

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11
Q

What is a prion?

A

An infectious misfolded protein which causes other proteins of the same kind to misfold

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12
Q

What is the composition of a protein?

A

Made up of carbon, hydrogen, oxygen, nitrogen (12-18%) & ~1% sulphur
>Molecular mass of 10,000-1 million g/mol

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13
Q

What percentage of albumin is made up of nitrogen?

A

16%

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14
Q

Cellular role of protein?

A

Transport & storage of other molecules, structural framework, antibodies & blood clotting factors, enzymes

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15
Q

What role do proteins play in support?

A

Collagen in ligaments, tendons & skin
Keratin in hair & nails

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16
Q

What are enzymes

A

Majority are proteins. Catalyst speed up chemical reactions.

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17
Q

What role do proteins play in transport?

A

Haemoglobin in blood, membrane proteins

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18
Q

What role do proteins play in immunity?

A

Antibodies are proteins

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19
Q

What role do proteins play in regulation?

A

Insulin - regulates blood sugar

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20
Q

What role do proteins play in motion?

A

Contractile proteins e.g. actin & myosin cause muscle movement

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21
Q

Reference range in recumbent patients

A

62-82 g/L
Proteins make up ~7% of the mass of serum (91% solvent & 2% nutrients)

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22
Q

What method is used generally to measure total protein level?

A

Colorimetry

23
Q

Explain the Kjeldahl Titration method

A

> 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

24
Q

Explain the Biuret method

A

> 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

25
Q

Explain the Phenol Method

A

> 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

26
Q

What is the Folin-Lowry method?

A

A mix of Biuret and Phenol method - 100x more sensitive than Biuret alone

27
Q

Explain the Turbidimetric & Nephelometric Method?

A

> 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

28
Q

Explain Refractometry

A

> 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

29
Q

Determination of Total Protein in Urine

A

> Biuret method - acid precipitated proteins/concentrate from membrane filtration - equally sensitive to each protein
Turbidimetric & dye binding methods - less time consuming

30
Q

Reference range of spot urine

A

<140mg/24 hours

31
Q

Determination of Total Protein in Cerebrospinal Fluid

A

> Turbidimetric methods & Coomassie Brilliant Blue dye-binding method (Bradford method)
Turbidimetric method - requires 0.2-0.5ml
CBB - as small as 25 ul

32
Q

CSF Proteins reference range

A

200-400 mg/L

33
Q

Elevated Proteins in CSF

A

Infectious, intracranial haemorrhages, multiple sclerosis, Guillian Barré syndrome, malignancies, endocrine abnormalities, certain medication, some inflammatory conditions

34
Q

Reduced proteins in CSF

A

Repeated lumbar punctures or chronic leak of CSF

35
Q

Components of an electrophoresis system

A

1) Support media
2) Power supply
3) Electrodes and chambers
4) Dryer
5) Visualisation
6) Densitometer

36
Q

Albumin Method

A

> 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

37
Q

Albumin reference range

A

35-52 g/L

38
Q

Explain Electrophoresis

A

> 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

39
Q

How are proteins and amino acids amphoteric?

A

Act either as proton donors (acid) & proton acceptors (base)

40
Q

How do proteins act in a basic solution?

A

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

41
Q

In protein electrophoresis, explain the support media

A

Usually cellulose acetate or agarose gel-coated plastic plates

42
Q

In protein electrophoresis, explain the power supply

A

> Provide a constant voltage (50 - 200V)
Higher voltage - improve resolution & decrease time taken - can heat up buffer & melt gel

43
Q

In protein electrophoresis, explain the electrodes and chambers

A

> 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

44
Q

In protein electrophoresis, explain visualisation

A

> 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

45
Q

In protein electrophoresis, explain the densitometer

A

> Spectrophotometer - measure light transmittance through a solid sample
The plate is positioned over the monochromator - can scan plates - plates moved across the exit slit

46
Q

What is the general procedure for electrophoresis?

A

> 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

47
Q

Order of bands in electrophoresis strip

A

1) Albumin
2) Alpha-1
3) Alpha-2
4) Beta
5) Gamma

48
Q

A note on albumin

A

> 50-60% total plasma proteins - main protein produced by liver
Maintain osmotic pressure
Carrier via reversible binding to bilirubin, lipids, hormones, metals, drugs

49
Q

A note on Alpha-1 globulins

A

> Main component alpha-1-antitrypsin (produced by liver - protects lungs)
Deficiency - liver disease & childhood pulmonary emphysema
Also include Glycoprotein and alpha-1-lipoprotein (HDL)

50
Q

A note on alpha-2-globulins

A

> main component alpha-2-macroglobulin (inhibit protease & act as carrier)
Increased in nephrotic syndrome
Include haptoglobulin & caeruloplasmin

51
Q

A note on beta-globulins

A

VLDL, transferrin, complement

52
Q

A note on Gamma globulins

A

Immunoglobulins

53
Q

Multiple myeloma in protein electrophoresis

A

An extra M band between beta and gamma globulins

1.1 Clinical Chemistry (7 decks)

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