assignment 6 Flashcards
what function does insulin regulate in the body
- regulates metabolism of carbs, proteins, fats
what are effects of high insulin concentration
- low blood glucose (glucose - glycogen)
what are effects of low insulin concentration
- high blood glucose (glycogen to glucose)
- which begins protein catabolism
what is basal concentration of insulin in a human
10^-10 M
how was insulin discovered
- Pancreatic ducts in dogs were tied off.
- This caused most pancreatic cells to die, leaving the Islets of Langerhans behind.
- Insulin extract was obtained from these.
What were the original sources of commercial animal-derived insulin?
- animals slaughtered for food
- harvest pancreas as by product of meat production
- cows, pigs, horses, fish
list common impurities found in animal source insulin
- proinsulin: allergenic, switch sources
- proteases: slowly destroy proteins, limit shelf life
- glucagon
- somatostatin
what is the general structure of insulin
- 2 peptide chains joined by disulphide bonds
- both chains in alpha helix conformation
how many amino acids in insulin
A chain has 21
B chain has 30
how many disulfide bridges in insulin
- 3
- 1 intrachain (A-A)
- 2 interchain for (A-B)
oligonucleotides made via SPS: why is it important to perform capping
- IF NOT CAP: OH will react in subsequent couplings
- creates mixture of several DNA sequences (desire and several deletion)
- mixture hard to purify as all DNA strands have similar properties
- by capping, prevent formation of other DNA chains
oligonucleotides made via SPS: how is the final deprotection and removal from resin performed
- Add NH3
- removes all PG from phosphate and DNA from resin
DNA polymerase enzymes add nucleotides using the 3’-OH, while DNA synthesizers add nucleotides using the 5’-OH. Why is the 3’-OH not used for machine-based synthesis?
- 5OH primary
- 3OH secondary
- primary better nucleophiles than secondary as less steric hindered
- better results in displacement chemical rxns by using best nucleophile
What does the method of making DNA mean for the researcher planning to synthesize DNA? (DNA SYNTHESIZER)
Nucleotides are added in the opposite order on a DNA synthesizer than they are by a polymerase enzyme.
What are the six basic steps to follow when making a recombinant protein?
- isolate the DNA for the protein you want to make
- insert the DNA into a vector
- insert the vector into cells
- isolate the cells that express the protein you want 5. grow these cells in quantity
- isolate and purify the protein
What are the two most common types of vectors used in recombinant work?
plasmids and viruses
What is a plasmid?
- Autonomously replicating minichromosome.
- A small piece of circular DNA found in bacteria.
What type of plasmid is preferred and why?
- episomes
- amplified copy numbers inside bacteria, higher amount of protein produced
general features found on plasmids
- antibiotic resistance gene
- colour producing enzymes
- polylinker
role of antibiotic resistance gene in plasmid
- used to purify transformed bacteria
- place vectors in bacteria, some bacteria take up vectors and some do not
- grow bacteria on medium containing antibiotic
- only those bacteria that take up bacteria vector survive
role of colour producing enzymes in plasmid
- to identify transformed cells
- only bacteria that take up and express vector produce colour
role of polylinker in plasmid
- special DNA sequence containing lots of restriction sites
- allows plasmid to be used with lots of restriction enzymes
What type of enzyme is used to cut DNA in recombination
- restriction enzymes
What are the two types of ends that are produced by restriction enzymes?
- blunt and sticky
Which type of end is most useful for recombinant work and how do these ends help DNA splicing?
- sticky end
- overhands make it easier for 2 DNA strands to join together
What type of enzyme is used to splice pieces of DNA together?
- ligase
What is the major issue associated with insulin administration that makes insulin NOT DRUG LIKE
- concentration levels between normal blood levels and commercial drug (10^-10 and 10^-3)
How is this issue of insulin NOT being drug like addressed by the method of administration
- subcutaneous injection into fat layer under skin, drug preciptates out and then slowly dissolves resulting in lower concentration
- injecting zinc crystals slows dissolution, zinc forms hexemers with insulin which lower rate of dissolution
Prior to 1985, how did the chemical properties of insulin help to solve this challenge?
- zinc crystals of insulin form hexemers which dissolve slowly in water
what is lispro
- first fast acting insulin to be developed
- switched order of 2 amino acids in insulin (Pro-Lys to Lys Pro)
- similar to that of IGF1 which dissolves quickly in water (similar amino acid sequence, Lys-Pro seq)
- disrupted oligemrization of zinc crystals, speeds up dissolution
what is aspart
- latest fast acting insulin
- proline changed to aspartic acid, changes neutral to negative charge
- charge repulsion between molecules in crystal speeds up dissolution
what is glargine
- first slow acting insulin
- B chain 2 arginines added to C terminal
- A chain: C terminal asparagine replaced by glycine
- arginine raises isoelectric point from 5.4 to 7.2
- new isoelectric is closer to 7.4 which makes less water soluble in physiological pH
what is detemir
- another slow acting insulin
- B chain: last amino acid was removed, attach palmitic acid to 2nd last amino acid
- reduced water solubility and increased lipid solubility, slower dissolution into blood
- MAINTAIN MORE CONSISTENT BASAL CONCENTRATION
what is HPLC
- high pressure liquid chromatography
- better quality separations
- uses solid phase with very small particles size, increases effective surface area of solid phase
- increases molecular interactions involved in separation
- requires high pressure to force liquid through small spaces between particles
