Plasma Proteins - Introduction and Methods of Analysis Flashcards
What percentage of whole blood is plasma
55%
What is plasma composed of?
Water (92%)
Proteins (4%)
Lipids (3%)
Other solutes (1%)
What proteins are found in plasma
(4)
60% albumin
35% globulins
4% fibrinogen
1% regulatory hormones
What does albumin do
(3)
Contributes to plasma osmotic pressure
Binds to hormones and inactivates them
Transports lipids
What do globulins do?
Transport ions, hormones, immune function
What does fibrinogen do
Essential component of clotting system
What are regulatory proteins
Enzymes and hormones
How many plasma proteins are there
150+
What is the range of sizes in plasma proteins
Between 21 kDa and 971 KDa
What is plasma vs serum
Plasma = anticoagulant added - contains all clotting proteins
Serum = allowed to clot - clotting removes many proteins especially fibrinogen so the total protein concentration will be lower
What is the role of plasma proteins
(4)
They are trapped in the vascular system -> cant fit in capillaries
They provide a colloidal osmotic pressure
They maintain normal blood volume
Maintain normal water load in interstitial fluid and tissues
What is the intracellular compartment
Consists of the fluid inside of cells
What is the extracellular compartment
Consists of the interstitial fluid and intravascular fluid (plasma)
How is total body water distributed
2/3 is in the intracellular fluid compartment
1/3 in the extracellular fluid compartment
What makes up extracellular fluid
75% interstitial fluid
25% intravascular fluid/plasma
What proteins are responsible for transport/binding
Albumin
Apolipoprotein
Haptoglobin
Thyroxine binding protein
What protein is responsible for oncotic pressure
Albumin
List some enzyme inhibitor proteins
(3)
Alpha-1-antitrypsin
Cystatin C
C 1-esterase inhibitor
List three immune defence proteins
Immunoglobulins
Complement
C-reactive protein
List three acute phase proteins
C-reactive protein
Alpha 1 acid-glycoprotein
Serum amyloid A (SAA)
List some proteins with enzymatic activity
(5)
Complement C3
Complement C4
Haptoglobin
Cerulopasmin (CER)
Alpha-1-antitrypsin
List some tissue derived proteins
(5)
Fibrinogen
Antithrombin
Coagulation factors
Proteins of the fibrinolysis process
Fibronectin
Where are most proteins synthesised
The liver
Where are gamma globulins synthesised
Synthesised and secreted by plasma cells
Where are plasma proteins synthesised
(3)
By the liver cells
By endothelial cells
By blood cells e.g. lymphocytes and plasma cells (immunoglobulins)
What influences protein synthesis
(4)
Nutritional status
Various feedback mechanisms
Hormonal factors
Genetic factors
How much albumin do you make a day
About 10g of albumin
Where does degradation of proteins occur
(2)
Mainly in the liver
Some proteins e.g. albumin are eliminated via the renal system
How are proteins broken down in the liver
(3)
Proteins are endocytosed by hepatocytes
Plasma proteins are then deglycosylated and then cleaved into amino acids by proteinases and peptidases
This occurs in the lysosomes and cytosol of the cell
Where in hepatocytes does protein breakdown occur
Lysosomes and the cytosol
Describe protein homeostasis
(2)
Synthesis of protein must be equal to protein catabolism
i.e. protein production must equal protein loss
What affects protein concentrations in blood
(2)
Abnormalities -> which arise from a wide variety of unrelated conditions
Alterations in hydration which affect the concentration of proteins but not the absolute amount
List the most important groups of proteins
(11)
Enzymes
Polypeptide hormones
Antibodies and complement components
Plasma proteins
Apolipoproteins
Haemoglobin
Protein coagulation factors
Structural proteins
Contractile proteins
Storage proteins
Chromosomal proteins
What is the main role of polypeptide hormones
They regulate metabolism
What is the main role of plasma proteins
Maintain oncotic pressure
Name three structural proteins
Keratin
Collagen
Elastin
Name a contractile protein
Myosin
Name a storage protein
Ferritin
Name a chromosomal protein
Basic histones
What is the equation for total plasma proteins
Albumin + globulins + fibrinogen
What is the equation for globulins
Total proteins-albumin = globulin level
Why do we measure total protein and globulins
They give us a general index of overall health and nutrition
The globulins also contain antibodies
What is the albumin globulin ration and why do we measure it
(2)
Serum albumin/ serum globulin g/l
This ratio can help us identify disorders of serum proteins
What is the optimal albumin/globulin ratio
1.5 -> 2.2
List the 6 methods used to analyse total proteins
Kjedahl method
Biuret reaction
Dye binding reactions
UV spectrophotometry
Refractometry
Turbidometric methods
What is the Kjedahl Method
(5)
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
(2)
Most common method used to analyse protein
It is an automated process in labs
What is the principle behind the Biuret reaction
(3)
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
What are the downfalls of the Biuret Reaction
(2)
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 dye binding methods are used to analyse proteins
(3)
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
What are the downfalls of dye binding methods to analyse proteins
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 dye is specifically used for albumin
Bromocresol green
How can UV spectrophotometry be used to analyse proteins
(4)
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)
What are the downfalls of UV spectrophotometry to analyse protein
(2)
Difficult to standardise for complex protein mixtures
Only used for specific proteins such as albumin or IgG
Give two examples of aromatic amino acids
Phenylalanine and tyrosine
What is refractometry used for in the analysis of proteins
Used for estimating plasma protein (including fibrinogen) in EDTA plasma
What is the principle of refractometry
(2)
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
What is the main downfall of refractometry
Lipaemia and moderate to severe haemolysis renders the results invalid
What is the turbidometric method of analysing protein
(2)
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
What is the principle behind turbidometry
The process of measuring the loss of intensity of transmitted light due to the scattering effect of particles suspended in a sample
How sensitive is turbidometry
Sensitive to as little as 60 mg/L of protein
