Protein Flashcards
What are the six different methods of measuring proteins
Kjedahl method
Biuret reaction
Dye Binding Methods
UV Spectrophotometry
Refractometry
Turbidometric
What is the Kjedahl method
This measures nitrogen. We assume that protein contains 16% nitrogen.
Any nitrogen containing compounds (proteins) in plasma are digested with sulphuric acid, a catalyst and a salt.
Ammonia is produced which is then distilled into a boric acid solution and titrated with acid.
Must then correct for non-protein nitrogen by analysing a protein free filtrate as well
What is the Biuret reaction
Most common method used to analyse protein. It is an automated process in labs.
Copper reacts in an alkaline solution with peptide linkages to produce a violet coloured complex.
This can be measured used a spectrophotometer at absorbance 540 nm.
This detects all different types of proteins and is accurate for the range of 10-100g/l.
Need at least 2 peptide linkages -> will not detect free amino acids.
Not sensitive enough for low concentrations i.e. can’t be used for CSF, urine and many body cavity effusions
What is the dye binding methods
Proteins can bind certain dyes resulting in a change of absorption spectrum.
Coomassie blue and pyrogallol red are typically used.
Different proteins bind different amounts of due therefore the colour change depends on the composition of the protein.
Difficult to standardise for complex protein mixtures as each protein absorbs a different amount of dye.
Some dyes are specific e.g. bromocresol green for albumin
What is UV Spectrophotometry
Aromatic amino acids such as phenylalanine and tyrosine absorb light at 280nm.
Therefore abs 280 can be used to estimate protein composition e.g. 1g/L Albumin = Abs280 of 0.530.
Therefore if your sample reads at 0.530 it contains 1g/L albumin.
By knowing the Molar absorptivity of a particular protein its concentration can be determined using Beers Law (A = ebc).
Difficult to standardise for complex protein mixtures.
Only used for specific proteins such as albumin or IgG
What is refractometry
Measure the refractive index of a sample relative to the refractive index of water.
The reading is a measurement of total solids and is only an estimate of protein concentration since there is variation in other serum components e.g. sodium, phosphate, glucose etc which can affect the refractive index as well.
Lipaemia and moderate to severe haemolysis renders the results invalid.
Used for estimating plasma protein (including fibrinogen) in EDTA plasma.
What is turbidometric
A sensitive method of quantifying protein in low-protein fluids such as CSF or urine.
Used when protein level is too low for biuret or refractometer method.
The principle is based on the process of measuring the loss of intensity of transmitted light due to the scattering effect of particles suspended in a sample.
Turbidometry is sensitive to as little as 60mg/L of protein.
Write about albumin
Albumin is a highly polar, highly soluble protein synthesised by the liver using dietary protein.
Albumin comprises 35-50% of total blood protein i.e. it is the most abundant protein and has a half life of 15-19 days.
Albumin contributes to plasma osmotic pressure, binds to hormones and inactivates them and transports lipids.
Albumin’s presence in fluids creates an osmotic force that maintains fluid volume within he vascular space.
A person makes about 10g of albumin a day. Albumin is a very strong predictor of health.
It’s important that albumin is a highly soluble medium sized molecule as it has to be small enough to pass through fenestrated endothelium in the nephron.
Albumin also needs to be soluble in blood to transport fatty acids, cations and positively charged drugs.
The body needs to produce albumin daily as we have no storage form or reserve of albumin.
Albumin homeostasis is vital in the body i.e. albumin synthesis must equal albumin catabolism, loss and/or distribution.
A decrease in circulating albumin causes oedema.
Albumin concentration is determined by measuring the amount of intravascular albumin mass/plasma volume.
Hypoproteinaemia is almost always caused by hypoalbuminemia with the exception of Ig deficiencies.
We measure albumin usually to investigate the cause of hypoproteinaemia or hyperproteinaemia.
Hyperproteinaemia due to dehydration will show a high total protein and albumin level but hypoproteinaemia due to hypergammaglobulinemia will show a raised total protein with a normal/low albumin.
What might cause albumin levels to decrease
Malnutrition,
malabsorption,
advanced chronic liver disease, hypoalbuminemia or
analbuminemia,
increased catabolism,
increased loss e.g. in nephrotic syndrome, exudative loss in burns, haemorrhage, gut loss
increased redistribution e.g. haemodilution, increased capillary permeability (septicaemia) or decreased lymph clearance.
What is the usual cause of hypoproteinaemia
Hypoalbuminemia with the exception of Ig deficiencies.
What is the usual cause of Hyperproteinaemia
Hyperproteinaemia due to dehydration will show a high total protein and albumin level but hypoproteinaemia due to hypergammaglobulinemia will show a raised total protein with a normal/low albumin.
Explain CRP
C Reactive protein (CRP) is an immune defence protein and an acute phase protein i.e. levels of CRP become elevated during inflammation.
Since its an acute phase protein C Reactive protein is measured to investigate inflammation.
CRP has replaced the erythrocyte sedimentation rate to measure inflammation in bacterial infections and acute inflammatory conditions.
Slightly elevated levels of CRP indicate chronic, low grade inflammation and have been correlated with an increased risk of cardiovascular disease.
CRP is the most sensitive acute phase protein with levels increase several hundredfold in inflammation.
CRP has a half life of 17 hours, peak levels are usually reached within 48-72 hours and it’s clearance is as exponential as its production.
Persistent CRP beyond a week indicates continuation of a pathological process or a complication.
CRP is often used when screening healthy patients such as blood donors or outpatients as it is extremely sensitive.
CRP can also be used to monitor infectious diseases with the goal to minimise the use of antibiotics i.e. a low CRP level indicates a patient may not need antibiotics.
The highest CRP levels are seen in bacterial infections, lower levels are seen in fungal/parasitic infections and viral infections are less likely to cause substantial elevations.
CRP is often measured alongside procalcitonin for the early diagnosis of bacterial and mycotic infection as well as sepsis and multiple organ failure.
Write about AAT
Alpha-1-antitrypsin
An enzyme inhibitor protein. It is the most important proteinase inhibitor found in serum. It is synthesised by the liver.
It works by inactivating several serine proteases by irreversible forming an inhibitor complex.
There are various isoforms of AAT with the most common being PIMM. AAT works on proteases such as leukocyte elastase, trypsin, chymotrypsin, collagenase, plasmin and thrombin.
AAT is an acute phase reactant which is found elevated in inflammatory processes.
Elevated levels are often used to help diagnose inflammation of the liver parenchymal cells as this doesn’t raise other APRs.
Certain genetic conditions can cause a deficiency of AAT.
AAT also plays a role in protecting self cells from the work of white blood cells during infection.
When an individual lacking AAT suffers from an infection their own cells will also be attacked by chemicals produced by white blood cells, the elastin in lungs is particularly affected by this.
In those with genetic deficiencies of AAT it may be due to the AAT not being able to leave the liver and not due to a synthesis issue.
If this is the case, high levels of trapped AAT will cause liver damage.
Write about myoglobin
The haemoglobin equivalent found in muscle cells.
Allows marine animals such as whales to stay underwater for long periods of time.
Carries oxygen in muscle.
Myoglobin has a high affinity for oxygen, allowing it to readily bind to oxygen molecules.
When oxygen is abundant, myoglobin binds to oxygen molecules, effectively storing them within muscle cells.
This oxygen reserve can be utilized when oxygen supply is limited
Myoglobin is responsible for the reddish-brown color of muscle tissue.
When myoglobin is oxygenated, it has a bright red color, while when it is deoxygenated, it appears darker.
Myoglobin is released into the bloodstream when muscle cells are damaged or injured. Elevated levels of myoglobin in the blood can be detected and measured in diagnostic tests, such as serum myoglobin assays.
This can be useful in diagnosing and monitoring conditions involving muscle damage, such as myocardial infarction
