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biopharm > assignment 6 > Flashcards

assignment 6 Flashcards

1
Q

what function does insulin regulate in the body

A
  • regulates metabolism of carbs, proteins, fats
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2
Q

what are effects of high insulin concentration

A
  • low blood glucose (glucose - glycogen)
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3
Q

what are effects of low insulin concentration

A
  • high blood glucose (glycogen to glucose)
  • which begins protein catabolism
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4
Q

what is basal concentration of insulin in a human

A

10^-10 M

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5
Q

how was insulin discovered

A
  • 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.
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6
Q

What were the original sources of commercial animal-derived insulin?

A
  • animals slaughtered for food
  • harvest pancreas as by product of meat production
  • cows, pigs, horses, fish
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7
Q

list common impurities found in animal source insulin

A
  • proinsulin: allergenic, switch sources
  • proteases: slowly destroy proteins, limit shelf life
  • glucagon
  • somatostatin
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8
Q

what is the general structure of insulin

A
  • 2 peptide chains joined by disulphide bonds
  • both chains in alpha helix conformation
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9
Q

how many amino acids in insulin

A

A chain has 21
B chain has 30

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10
Q

how many disulfide bridges in insulin

A
  • 3
  • 1 intrachain (A-A)
  • 2 interchain for (A-B)
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11
Q

oligonucleotides made via SPS: why is it important to perform capping

A
  • 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
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12
Q

oligonucleotides made via SPS: how is the final deprotection and removal from resin performed

A
  • Add NH3
  • removes all PG from phosphate and DNA from resin
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13
Q

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?

A
  • 5OH primary
  • 3OH secondary
  • primary better nucleophiles than secondary as less steric hindered
  • better results in displacement chemical rxns by using best nucleophile
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14
Q

What does the method of making DNA mean for the researcher planning to synthesize DNA? (DNA SYNTHESIZER)

A

Nucleotides are added in the opposite order on a DNA synthesizer than they are by a polymerase enzyme.

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15
Q

What are the six basic steps to follow when making a recombinant protein?

A
  1. isolate the DNA for the protein you want to make
  2. insert the DNA into a vector
  3. insert the vector into cells
  4. isolate the cells that express the protein you want 5. grow these cells in quantity
  5. isolate and purify the protein
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16
Q

What are the two most common types of vectors used in recombinant work?

A

plasmids and viruses

17
Q

What is a plasmid?

A
  • Autonomously replicating minichromosome.
  • A small piece of circular DNA found in bacteria.
18
Q

What type of plasmid is preferred and why?

A
  • episomes
  • amplified copy numbers inside bacteria, higher amount of protein produced
19
Q

general features found on plasmids

A
  • antibiotic resistance gene
  • colour producing enzymes
  • polylinker
20
Q

role of antibiotic resistance gene in plasmid

A
  • 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
21
Q

role of colour producing enzymes in plasmid

A
  • to identify transformed cells
  • only bacteria that take up and express vector produce colour
22
Q

role of polylinker in plasmid

A
  • special DNA sequence containing lots of restriction sites
  • allows plasmid to be used with lots of restriction enzymes
23
Q

What type of enzyme is used to cut DNA in recombination

A
  • restriction enzymes
24
Q

What are the two types of ends that are produced by restriction enzymes?

A
  • blunt and sticky
25
Q

Which type of end is most useful for recombinant work and how do these ends help DNA splicing?

A
  • sticky end
  • overhands make it easier for 2 DNA strands to join together
26
Q

What type of enzyme is used to splice pieces of DNA together?

A
  • ligase
27
Q

What is the major issue associated with insulin administration that makes insulin NOT DRUG LIKE

A
  • concentration levels between normal blood levels and commercial drug (10^-10 and 10^-3)
28
Q

How is this issue of insulin NOT being drug like addressed by the method of administration

A
  • 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
29
Q

Prior to 1985, how did the chemical properties of insulin help to solve this challenge?

A
  • zinc crystals of insulin form hexemers which dissolve slowly in water
30
Q

what is lispro

A
  • 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
31
Q

what is aspart

A
  • latest fast acting insulin
  • proline changed to aspartic acid, changes neutral to negative charge
  • charge repulsion between molecules in crystal speeds up dissolution
32
Q

what is glargine

A
  • 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
33
Q

what is detemir

A
  • 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
34
Q

what is HPLC

A
  • 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

biopharm (8 decks)

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