MCP Flashcards
basic structure of myoglobin and hemoglobin
myoglobin: primary structure= 153 AA intracellular protein, secondary structure= 8 alpha-helices with a heme prosthetic group in a hydrophobic pocket, tertiary structure: similar to alpha and beta Hb subunits only longer
hemoglobin: globular tetrameric protein with the quaternary structure of a2b2 (dimer of ab protomers) arranged as a tetrahedron where each chain is bound to a heme (thus binds 4 O2 molecules= 8 oxygen atoms) and the two ab dimers switch back and forth between states T and R (15 degree rotation)
- ApoHb: a2b2 without prosthetic groups
- HoloHb: ApoHb + 4 hemes
cooperative O2 binding
reflects the existence of two Hb states (T and R) in which the more O2 bound to a tetramer, the greater the affinity is for binding another oxygen (what is happening at one site promotes the same things happening at another identical site- requires having multiple sites of the same kind)
T- low affinity
R- high affinity
(when equilibrium is affected by something other than oxygen, the steep region of the sigmoidal Hb oxygen dissociation curve shifts to the left or right)
the effect of pH, [CO2] and [BPG] on the O2 binding curve
pH= higher pH- O2 is loaded; lower pH- O2 is released
[CO2]= high CO2 lowers pH releasing O2; low CO2 increases pH loading O2
[BPG]= moves curve to right, stabilizing the T-state
sickle cell anemia
HbS occurs when there is a single substitution (Glu–>Val) at the beta 6 position leading to hemolytic anemia if individuals are homozygous for the gene causing aggregation and polymerization into rigid extended helical fibers and distortion of the RBC shape into sickles)
if heterozygous, Hb is 40% of its normal value leading to a shorter than normal lifetime for erythrocytes but a 73% reduced rate of contracting malaria (evolutionary selected protection)
thalassemia and possible treatments
insufficiency of alpha or beta chains leading to insufficient or non-functional hemoglobin. major= lack of chains (homozygous or compound heterozygous) and minor= decreased chains (simple heterozygous-carrier)
alpha-thalassemia: defect in alpha chain production; b4 or g4 (HbH in adults and HbBarts in fetuses)
beta-thalassemia: defect in beta chain production
*treatments: blood transfusion (temporary) or bone marrow transplant
methemoglobinemia
hemoglobin in which the heme iron has been oxidized to the ferric state preventing the binding or release of O2 caused by mutations that may stabilize the oxidized form, CYB5R defect or chemical agents such as nitrite
adaptive mechanisms to make up for insufficient diffusion
oxygen carriers (respiratory pigments: hemoglobin and myoglobin) and circulatory systems (respiratory fluid: blood) to speed up downhill diffusion from alveoli (100 torr) to capillaries in active tissues (20 torr)
*myoglobin: facilitates O2 diffusion to the mitochondrion within the muscle cell
protomer
the smallest subassembly with the same composition as the whole complex
example: ab in Hb (which is a2b2)
heme structure
Fe chelated by protoporphyrin IX- which is a porphyrin (tetrapyrrole bridged by methylene groups) attached to 4 methyl, 2 vinyl and 2 propionate substituent groups
Fe binds O2 and is hexacoordinate with 4 equatorial ligands bound to 4 nitrogen atoms facing the center of the structure and 2 axial ligands in front and behind of the heme plate
deoxy vs. oxyHb structure
deoxy: iron is pentacoordinate instead of hexacoordinate since there is an empty space on the distal side of the Fe (“distal heme pocket”) which is where O2 usually binds
oxy: Fe is hexacoordinate in which the O2 is bound between the Fe (II) and His E7 (distal His), through hydrogen bonding, which destabilizes an intermediate formed when O2 tries to oxidize Fe and lowers the affinity for CO
* has 5 nitrogens- 4 from pyrroles and 1 from the proximal His side chain
colors of blood when Hb is oxygenated or deoxygenated and when Hb and Mb is oxidized
ferro/Fe (II)/ferrous:
1. oxyHb: (O2 is present) brilliant scarlet (transmits more light in red region)
- deoxyHb: (no O2) dark red
ferri/Fe (III)/ferric:
1. oxidized Hb/Mb: yields metHb/metMb which is brown-red like dead meat
poisons that bind as the 6th ligand
Co, NO, H2S, and anions cyanide, azide, and sulfide (CN-, S-) bind as the 6th ligand and prevent O2 from binding making them toxic
oxygen dissociation curves for Mb and Hb
Mb: hyperbolic curve with a low P50 (2.6 torr) and binds O2 under conditions in which Hb releases it
Hb: sigmoidal curve with a high P50 (26 torr)
allostery
an effect of the same or different molecule binding at another site that is different
positive and negative effectors of Hb oxygen dissociation curve
positive effector: increases affinity (O2) shifting curve to the left thus stabilizes the R state
negative effector: decreases affinity and shifts the curve to the right thus stabilizing the T state
*increases the affinity for O2 in the lungs to promote full saturation and decreases the affinity in the tissues to promote unloading
BPG
negative effector that adjusts the overall average P50 to put the steepest part of the binding curve between the lung pO2 and the tissue pO2 changing midpoint affinity stabilizing the T state by cross-linking the b subunits thus decreases Hb’s oxygen affinity allowing it to be released (efficient O2 carrier)
*responds to long-term changes
acclimation to low O2 pressures
adapt decreased O2 concentration (Hb with P50 of 26 would not be saturated) by decreasing the affinity, to put the steepest part of the curve between alveolar and capillary pO2, by increasing BPG to squeeze more O2 out of the hemoglobin and into the tissues
*max oxygen consumed during strenuous exercise is significantly decreased
Bohr effect
- oxygenation of Hb makes it a stronger acid
- releasing 0.6 protons for each O2 bound
higher pH–> O2 is loaded
lower pH–> O2 is released
CO2 effect on O2 affinity in tissues and lungs
tissues: high CO2 will lower pH thus stabilizing the T state reducing the affinity for O2 promoting release
CO2 + H2O H2CO3 HCO3- + H+
lungs: low CO2 will increase pH thus stabilizing the R state increasing the affinity for O2 promoting loading
CO2 + H2O <– HCO3- + H+
carbamylation of HGb
CO2 combines reversibly with the N-terminal amino groups of blood proteins to form carbamates stabilizing the T-state (reducing affinity of Hb for O2)
R-NH2 + CO2 R-NH-COOH
H+ released from carbamates further reduces the affinity of OxyHb for O2 (via Bohr effect)
R-NH-COOH R-NH-COO- + H+
HbF
fetal hemoglobin in which the beta subunits are replaces with gamma subunits increasing the affinity for O2 and decreasing the affinity for BPG
*wild type and common variants of hemoglobin
WILD TYPE:
- HbA (a2b2)
- HbA2 (a2d2)
- HbF (a2g2)
VARIANTS:
1. HbS (a2b*2): Glu–>Val at B6 position causing sickle cell anemia
- HbC (a2b”2): Glu–>Lys at B6 position protecting against malaria but will not polymerize
- HbH (b4): binds O2 very tightly and non-cooperatively hence providing inefficient oxygen delivery to tissues; arises when there are insufficient alpha chains as in alpha thalassemia major
- HbBarts (g4): in a fetus and is like HbH in which it binds O2 very tightly and non-cooperatively thus has poor oxygen delivery properties; arises when there are insufficient alpha chains as in alpha thalassemia major
CYB5R
*Cytochrome b5 reductase
- maintains a low level of hemoglobin in the MetHb form by reducing heme so it is converted back to its functional form
- mutation could cause methemoglobinemia in which CYB5R cannot compensate for the increasing amount of oxidized heme leading to cyanosis
gel electrophoresis of HbS vs. HbA
movement from negative/cathode to positive/anode side so the more negative hemoglobin will travel the furthest and fastest
HbA is more negative than HbS so it will travel further in a non-denaturing gel electrophoresis
*pulse oximeter
measures blood oxygen levels (N= 95-100%) by recording the amount of light passing through the finger at two different wavelengths around 650/red and 950/infrared since the absorbance of deoxy- and oxy- hemoglobin are very different (more deoxy at 650 and more oxy at 950)
*using change in absorbance takes into account the variation of pressure/volume of blood in the arteries during/between heart beats
pathway for heme synthesis and locations it occurs in
synthesis: Shemin pathway with Gly and succinyl CoA as precursors
locations: liver and erythroid cells (85%)
regulation of heme synthesis and how it could go wrong
erythroid cells: synthesize heme one time and heme stimulates heme synthesis
liver: heme is needed in varying amounts thoughout the liver cell’s lifetime so synthesis is tightly controlled
* main target of control is the first committed step which uses the enzyme ALAS which is feedback inhibited by heme through several pathways
porphyrin
oxidized tetrapyrrole forming metal chelates with a variety of metal ions which is photoreactive thus emits free oxygen radicals when exposed to light
protoporphyrin IX
isomer of protopoyrphyrin which has 8 side chains modified with three different groups in the order of MVMVMPPM
*this is found in heme and thus is chelated with Fe2+ (if chelated with Fe3+, it is referred to as hemin)
heme a, b and c and where they are found
- heme a- vinyl 2 is modified
- heme b- no modifications (MVMVMPPM)
- heme c- the vinyls are covalently bound to Cys residues of proteins
lead poisoning’s effect on heme biosynthesis
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porphyrias and their different types
porphyria: genetic deficiencies in heme metabolism in which enzymes are PARTIALLY defective leading to variable function
- different types:
1. hepatic-liver heme synthesis; induced
- erythropoietic- erythrocyte heme synthesis; chronic
- congenital erythropoietic- autosomal recessive uroporphyrinogen III co-synthase deficiency leading to the accumulation of uroporphyrinogen I and coproporphyrinogen I causing anemia and photosensitive skin
- protophorphyria- autosomal dominant partial deficiency in ferrochelatase leading to milder symptoms compared to congenital erythropoietic porphyria
- acute intermittent (liver)- autosomal dominant partial deficiency of porphobilinogen (PBG) deaminase leading to an accumulation of d-aminolevulinate and porphobilinogen causing a disease state when chemicals are introduced
- porphyria cutanea tarda (liver)- sporatic or autosomal dominant deficiency in uroporphyrinogen decarboxylase which is asymptomatic until liver is affected by ethanol or contraceptive pill
how drugs effect heme synthesis
heme synthesis is induced by toxins (ex: Tylenol) that also induce cytochrome P450 synthesis which is responsible for detoxification in the liver
cytochrome P450
oxidative enzymes involved in detoxification in liver in which heme is the prosthetic group
pathways of ALAS feedback inhibition
- repression of mRNA synthesis
- inhibition of translation of ALAS mRNA
- inhibition of transport of ALAS to mitochondria
- inhibition of enzyme
*ways for heme to control the amount of heme produced
how many Gly and succinyl CoA molecules due it take to make one heme molecules?
8 of each
step 1 of heme synthesis
Gly + succinyl CoA –(ALAS)–> ALA
- cofactor= pyridoxal phosphate
- location= mitochondria
step 2 of heme synthesis
2 ALA –(ALA dehydrase)–> porphobilinogen
- cofactor= Zn
- location= cytosol
- inhibitor= Pb which can replace Zn-cofactor (lead poisoning at even low levels)
*ALA looks like GABA (gamma-aminobutyric acid) and its accumulation leads to problems of lead posioning
step 3 of heme synthesis
4 porphobilinogen –(uroporphyrinogen synthase)–>uroporphyrinogen I
uroporphyrinogen I –(uroporphyrinogen III cosynthase)–> uroporphyrinogen III
- requires two enzymes: uroporphyrinogen synthase (catalyzes condensation and cyclization to form tetrapyrrole ring releasing 4 NH3) and uroporphyrinogen III cosynthase (catalyzes isomerization flipping D-ring for proper group orientation- switches A and P on one side of tetrapyrrole)
- location: cytosol
step 4 of heme synthesis
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step 5 of heme synthesis
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