Fundamentals of Pathology
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Name 3 features of the structure of RBCs
Biconcave disc no nucleus dont contain DNA,RNA or mitochondria
what are RBCs major constituent
Haemoglobin
What is the average life span of RBCs
120 days
How many polypeptide chains does Haemoglobin have, and what type
4 polypeptide chains (2 alpha globin and 2 beta globin chains)
How many haem groups per globin chain
1
What atom does Haem carry
Iron (Fe 2+)
What part of the amino acid determines the charge of the protein
The R group
Define Electrophoresis
The migration of charged particles in an electric field
Name factors which affect migration
Size, shape and charge of the particle current resistance
What is electrophoresis used for
For the separation/Purification of macromolecules
Name the 2 types of electrophoretic apparatus and when it’s used
Horizontal - agarose gel Vertical - polyacrylamide gel
How do you decide what apparatus to use
Uniform electric field across the gel Cooling system to prevent the thermal artefact Access to gel loading and monitoring
How is agarose gel prepared and when is it used
Agarose is a polysaccharide extracted from seaweed and is prepared by dissolving agarose in the buffer(heat and pour into casting tray) Used to separate large proteins(>200kDa)
How is polyacrylamide gel prepared and when is it used
Formed from the synthetic small molecule acrylamide and polymerises into long chains in the presence of a catalyst and initiator (APS and TEMED) Used in protein electrophoresis
What is a continuous buffer system
Uses same buffer in gel, sample and tank
What is a discontinuous buffer system
Different buffers used
What does migration of any protein in an electric field depend on
pl - constant given to any protein pH - determines the charge of the molecule
What is the electrophoretic technique used for separation
SDS-PAGE
Why is SDS used
SDS has a strong anionic detergent: to solubilise, dissociate and denature proteins to single chains disrupts H bonds and blocks hydrophobic interactions
Name the migration detection method
Protein staining - Coomassie brilliant blue
Factors affecting drug distribution
Blood flow/rate of presentation of the drug to the tissues Membrane transfer/fat: physicochemical properties e.g pH Binding to plasma membranes/tissue components
What is volume of distribution
The ‘apparent’ volume in which the drug is distributed
How do you calculate VD
VD=amount of drug in the body/[drug]plasma
Name 2 factors that affect the apparent VD of a drug and how
Drug-protein binding decreases Drug binding to tissues increases