summer BICH exam 1

how many oxygens do myoglobin bingd?

one molecule Oxygen

how many oxygens foes hemoglobin bind ?

4 molecules of oxygen

where is myoglobin located ?

in the muscle

what is the function of myoglobin ?

oxygen storage, binds O2 when not needed by tissues, releases it when necessary

what is the structure of myoglobin consists of ?

single polypeptide chain with 1 heme ground

what is the shape of myoglobin O2 binding curve ?

hyperbolic

what does a hyperbolic shaped curve indicate ?

high affinity, doesn’t easily give up oxygen

where is hemoglobin located at ?

red blood cells

what is the function of hemoglobin

transport of oxygen from lungs to tissue, binds O2 in lungs nad releases it in tissue

what is the structure of hemoglobin ?

tetramer and 4 heme group

what does deoxyhemoglobin mean

it menas there is low affinity and it has a low oxygen state

what does oxyhemoglobin mean

high affinity, saturated with oxygen

what is a heme

a prosthetic group with a protoporphyring ring and a ferrous iron that binds O2 in Mb and Hb

what is Myoglobin heme group ?

protoporphyrin ring with ferrous iron bound

what is structure like?

spherical ( globular), contains 8 helices, bends between helices

what is the role of iron in heme

Iron ( fe2+) binds oxygen and is coordinated by 4 nitrogens of the porphyrin rings, one from proximal His, and optionally one from oxygen

what type of proteins are Mb and Hb?

globular proteins that reversibly bind oxygen

what is the hill coefficeint (n) for Mb?

n=1 ( non-cooperative binding)

how many subunits and heme groups does Hb have ?

4 subunits ( 2 a, 2b) and 4 heme groups

what is the Hill coefficeint (n) for Hb?

n=3 ( positive cooperativity)

what is p50?

the partial pressure O2 at which the protein is 50% saturated

what does a lower p50 mean ?

higher affinity for oxygen

which has lower p50 Mb or Hb?

Mb ( Mb=2.8 torr , Hb=26torr )

what is Mb p50 value ?

2.8 torr

what is Hb P50 value ?

26 torr

what is the dissociation constant (kd)

the concentration of ligand at which half of the proteins binding site are occupied

what is the relationship between Kd and affinity ?

inverse- lower Kd= higher affinity

why is CO dangerous >

Co binds to heem 200x stronger than O2 blocking oxygen binding

what structural feature of Mb/Hb helps reduce CO binding ?

distal histidine ( His E7) causes steric hindrance, favoring angled binding of O2

what is the Hill equation used >

describes cooperative binding and helps calculate P50 and the Hill coefficient (n)

if N>1 in the Hill equation, what does that indicate?

positive cooperatively ( ie Hb)

what is the order of event based on structural changes occuring for oxygen binding in the first subunit of Hb?

1. movement of iron into the pane of the protoporhyrin ring

2. HisF8 relocates

3. movement of helix F

4. breaking of hydrophilic between dimers

5. 15* rotation collapse of central cavity

what does Hb do in the lungs?

releases CO2, binds O2, blood buffering system favors production of H2CO3.

what are the 3 types of restriction enzymes

type 1, type 2, type 3

what is the type 1 enzyme

requires ATP, non specific cleavage

what is type 2 enzyme

no atp, specific cleavage

what is type 3 enzyme

required ATP, specific cleavage

what is mean by sticky or blunt ends

they are formed when the palindromic sequence of DNA is recognized, and when cutting we are left with overhangs of DNA.

what is sticky ends ?

are sticky because they can be used to insert into plasmids for cloning vector purposes

what are blunt ends

when restriction enzymes cut both strands down the same line ( more applicable for analysis purposes)

why is DNA separated by size ( and not charge) during gel electrophoresis ?

charge is not applicable because the backbone of DNA is already negatively charged; meaning all start from the same negative charge status when electril field is applied

why does Chargaff’s rule (a=t and c=g) make sense chemically ? does this apply to base stacking ?

it makes sense because the nucleotide bases pair with each other through specific hydrogen bonds and compatible shapes ( sterics). these pairings are favorable and cause dna to be stable. base stacking is separate due to dimer dimer interactions between c-g bases using van der-waals forces.

what is the point of DNA forming a helix?

to minimize the electrostatic repulsion of the phosphodiester backbone

what three points of structure contribute to DNA stability?

hydrogen bonding between bases, electrostatic repulsion, and base-pair stacking interactions

what are the three intercalating agent discussed in class?

Ethidium Bromide, Acridine Orange, actinomycin D

what is the role Ethidium Bromide ?

aids in visualization in gel electrophoresis

what is the role of Acridine Orange ?

helpful for staining and identifying cells in certain cell cycle stages

what is the function of actinomycin D ?

antibiotic and prevents transcription

denaturing occurs during certain types of temps. what temperatures allow for denaturation for DNA, how would we renature the DNA, and what is the hyper chromic effect in context of DNA denaturation

hot temps denature, cool temps renature. the hyperchromic effect occurs during denaturation and the base are more accessible, causing the absorbance of new wavelengths of light

can we infer rations of nitrogenous bases from Tm or needed energy ? How?

yes we can, higher Tm indicate more G-C content due to H-bonding prevalence

what is the rate limiting step in the reannealing process?

binding -once the primer or appropriate sequence is found ( nucleation), chemistry or the annealing process happens almost instantaneously.

what are the steps of restriction mapping ?

-cut DNA with multiple restriction enzymes

-analyze fragments on gel

-map fragment together

what is the purpose of using probes in DNA visualization ?

to selectively illuminate regions of DNA using selective primers that are radioactive and emit fluorescence

What is the purpose of using ddNTPs in DNA sequencing

ddNTP are used to terminate DNA chain elongation. they help identify the DNA sequence by halting synthesis at specific nucleotides

why do ddNTPs stop DNA synthesis ?

ddNTPs lack both the 2= and 3- hydroxyl groups which causes termination of the chain

how do ddNTPS help determine DNA sequence?

ddNTPs are added at random during synthesis. each termination event creates fragments. when separated by gel, the fragment size reveals the nucleotide order.

what is a cloning vector

plasmids that are genetically modified to carry certain DNA. This allows for insertion of DNA within a genome, and for cloning purposes.

how is a cloning vector made?

• cut plasmid and foreign DnA with the same restriction enzyme

• insert foreign DNA into plasmid

• ligate the plasmid

• tranform a host cell with the plasmid

• host cell replicated the plasmid making a clone DNA

what are examples of cloning vectors and their size capacities ?

-plasmids ( up to 10kb)

-Bacteriophage ( lambda phage) ( up to 16 kB)

BACs Bacterial artificial chromosomes ( upto 300 kb)

what is the purpose of the selectable marker in a plasmid ?

it allows identification of bacteria that took up the plasmid. Most commonly an ANTIBIOTIC RESISTANCE gene

how does color screening with b-galactosidase help identify successful DNA

if foreign DNA is inserted into the b-gala gene region, the gene disrupted :

blue colonies= no insertion ( enzyme is active)

white/clear colonies = successful insertion ( enzyme disrupted )

what are the role of the ORI

allows the plasmid to replicate in the host cell

what is the role of RE ( restriction enzymes)

enable targeted insertion of foreign DNA at specific locations in the plasmid

explain the difference between forward and reverse primers

forward : matches 5-3 template strand = is comp and binds 3-5

reverse: matches 3-5 at end of DNA= binds to 5 to 3

what is purpose of PCR ( polymerase chain reaction)

is used to amplify a specific segment DNA, producing millions of copies for analysis

what do you need for PCR

template, primer, heat stable polymerase, 4 dNTPs for elongation

What are the steps of PCR

1. denaturation (high heat; separate DNA strands)

2. Annealing ( cool; allow primers to bind to target sequence )

3. Elongation ( warm; DNA polymerase to extend the DNA strand)

what does low PH indicate with blood

t state stabilization ( low O2 affinity)

what does high PH indicate with blood

r state stabilization ( high O2 affinity)

what is the role of blood buffering systems (BbS) in Hb oxygen binding?

the BBS regulates pH via CO2 and bicarbonate. in tissues :

• CO2 forms bicarbonate vis carbonic anhydrase, releasing H+

• this lowers PH, stabilizing t state and promoting O2 release

how does CO2 directly affect Hb T state ?

CO2 can bind allosterically to Hb forming carbamates on N terminal residues, this stabilizes the t state, helping unload oxygen in tissues