Myoglobin+and+Hemoglobin+Part+1


 * 06 September 2006**
 * Myoglobin and Hemoglobin**
 * Dr. John David Dignam, Ph.D.**

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=**Functions of Myoglobin and Hemoglobin**=


 * Proteins with similar functions, structure, and origins
 * Mb = myoglobin
 * Solubility of O2 in water is 35 μM
 * Not very soluble, and requires a carrier
 * Mb increases O2 available in muscle
 * [Mb] in red muscle = 500 μM (8g/L)
 * Capacity of O2 = 500 μM
 * That’s over 10 times O2’s solubility in water
 * Hb = hemoglobin
 * Hemoglobin carries O2 from capillaries in lungs to capillaries in lungs to capillaries in tissues
 * [Hb] in red blood cell = 4,000 μM (300g/L)
 * Capacity for O2 = 16,000 μM
 * Increases effective solubility of O2 by a lot!
 * Almost all vertebrates and invertebrates have hemoglobin
 * //Champsocephalus gunnari//, the ice fish, has no hemoglobin

=**Heme**=


 * Contains Fe-protophyrin IX ring
 * Carbon ring with 4 nitrogen atoms bound to Fe
 * Oxidized Fe+++ will not bind oxygen
 * Reduced Fe++ binds oxygen
 * Systems in blood maintain iron in Fe++ state

**Myoglobin**

 * Monomer
 * Single heme which is the O2 binding site
 * Single O2 site dominated by α helix
 * 80-85% α helix
 * Heme is held between 2 histidines and some hydrophobic residues
 * Help normally by hydrophobic interactions
 * Not covalently attached so if Mb is denatured, the heme falls off
 * O2 binds partly directly to iron and partly with one of the histidines

**Hemoglobin**

 * Essentially looks like 4 subunits which looks like myoglobin
 * Tetramer of (α2β2)
 * Can be dissociated into dimers
 * Four oxygen binding sites
 * Effector sites: protons, 2,3-BPG, CO¬2
 * 2,3-bis-phosphoglyceride regulates O2 affinity
 * Interactions holding heme to hemoglobin is mostly hydrophobic, just like Mb

=**O2 Saturation for Myoglobin**=


 * Calculate O2 concentration by adjusting partial pressure
 * Y = fractional saturuation
 * Y = 1 means all of it is saturated
 * Myoglobin has a hyperbolic binding pattern
 * Bound oxygen binds quickly at first, but eventually saturates
 * Protein-Ligand <--> P + L
 * Kd = Dissociation Constant
 * Kd = ( [P] x [L] ) / [PL]
 * [Pt] = [P] + [PL]
 * [PL] = ( [Pt] x [L] ) / ( Kd + [L] )

**Binding Isotherms**

 * Ligand and acceptor form a complex in equilibrium with free acceptor and ligand
 * Concentration of the complex depends on concentration of ligand and equilibrium constant (Kd)

**Fractional Saturuation**

 * Y = [PL] / [Pt]
 * Y = L / ( Kd + [L] )
 * Effect of [O2] on Y
 * Y = [O2] / ( Kd + [O2] )
 * When O2 equals 0, Y = 0
 * When O2 equals Kd, Y = 0.5
 * 1/2 Vmax
 * When O2 >>> Kd, Y approaches 1

**Determining Kd**

 * Determine [PL] at several concentrations of [L]
 * Use graphic or computer method to estimate Kd
 * Several graphic methods
 * Hyperbolic saturation plots
 * Linear transformations of binding equation
 * Apply linear or non-linear least squares fit of function to data to get estimates of Kd

**Scatchard Plot**

 * Y = mx + b
 * ( [PL] / [L]) = ( -1 / Kd ) x [PL] + ( [Pt] / Kd )
 * Y = [PL] / [L]
 * m = ( -1 / Kd)
 * X = [PL]
 * b = ( [Pt] / Kd )

**Double Reciprocal Equation**

 * Y = mx + b
 * ( 1 / [PL] ) = ( Kd / [Pt] ) x (1 / [L] ) + (1 / [Pt] )
 * Y = ( 1 / [PL] )
 * m = ( Kd / [Pt] )
 * X = (1 / [L] )
 * b= (1 / [Pt] )

=**Multiple Binding Sites**=


 * PL <--> P+L
 * K1 = [P] x [L] / [PL]
 * PL2 <--> PL + L
 * K2 = [PL] x [L] / [PL2]
 * If sites are independent and non-interacting, K1 = K2
 * If there is positive cooperativity, K1 > K2
 * If there is negative cooperativity, K1 < K2
 * Lower affinity with K is higher

**Adair Equation**

 * Y = Ka[X] + KaKb[X]2 / 1+ 2Ka[X]+Ka¬Kb[X]2
 * Protein with 2 binding sites where Ka and Kb are association constants for two different sites
 * Association constant is the inverse of a dissociation constant

=**Cooperativity and Allosterism**=


 * Hill approximation for ligand binding to multiple sites
 * //n// is the Hill coefficient corresponding to the minimum number of sites
 * Y = [L]//n// / K//n// + [L]//n//
 * Or, alternatively, Y = [L]//n// / K’ + [L]//n//

**Properties of Hill Equation**

 * Equation assumes only unliganded and fully liganded protein
 * //n// = Hill coefficient, does not need to be an integer
 * Determination of //n// gives measure of cooperativitiy
 * n=1, no cooperativity
 * e.g. myoglobin
 * n>1, positive cooperativity
 * Ligand binding at the first site makes binding at the second site easier
 * Characterized as sigmoidal
 * n<1, negative cooperativity
 * Ligand binding at the first site makes binding at the second site more difficult

**Hill Equation in Linear Form**

 * Y = [O2]//n// / Kdn + [O2]//n//
 * Log Y / (1-Y) = n Log [O2] – n Log Kd
 * Y = Log Y
 * m = n
 * X = Log [O2]
 * b = -n Log Kd

**Myoglobin vs. Hemoglobin**

 * Mb n = 1
 * Mb shows no cooperativity
 * O2 binds tightly and retained by Mb
 * Parabolic curve
 * Hb n = 2.8
 * Hb shows strong cooperativity
 * Hb doesn’t have high binding affinity for O2 because it needs to take up and eventually release O2
 * Sigmoidal curve

=**Monod Model**=


 * Concerted model for positive coopertivity
 * Hb has two conformational states:
 * R – relaxed, high affinity state
 * T – Taut, low affinity state
 * Has a cavity where a ligand can bind and stabilize the low affinity T state
 * Binds 2, 3 BPG
 * Conformers are symmetric (T4 or R4)
 * All subunits either in T or R state
 * Symmetry due to subunit contacts
 * Change the conformation of one subunit forces change in conformation of all to retain shape of Hb
 * Low affinity T and high affinity R states exist in equilibrium

**R and T States**

 * Both R and T states can bind O2
 * Specific subunit contacts stabilizes T state
 * O2 binding disrupts contacts in T state and promotes the formation of the R state
 * Low O2 favors the T state
 * Distribution of R and T states exist in the absence of O2; O2 just changes the distribution
 * O2 binding must be symmetrical – either none or all subunits are bound

=**Koshland Sequential Model**=


 * Absence of ligand, protein is in one conformation
 * Equalibrium of 2 states, R and T, but not present without the ligand
 * Symmetry is no required (RT states can occur)
 * Sequential changes in conformation
 * Ligand binding induces T --> R transition
 * Ligand subunit can influence the conformation of its neighboring subunit
 * Influence affinity for ligand to increase or decrease in affinity
 * Can account for both positive and negative behavior
 * Monod model cannot account for negative behavior easily

=**O2-Induced Structural Changes**=


 * Changes occur after the first 2 sites of Hb are bound
 * Fe moves 0.06 nm into the plane of the heme
 * Heme Iron is displaced into the ring of the heme by O2 binding
 * His on F8 helix moves with the iron
 * C=O of Val FG5 looses H-bonding contact with Tyr HC2
 * C-terminus is displaced
 * Diminished subunit interactions
 * Alters ionic interactions through salt bridges
 * Networks of ionic interactions is altered
 * Structural changes result in the cooperative nature of O2 binding

**Conserved Residues in Hb**

 * His Fe
 * Heme contact (proximal His)
 * His E7
 * O2 binding site (distal His)
 * Phen CD1
 * Heme contact
 * Leu F4
 * Heme contact
 * Gly B6
 * Allows B and E helices to contact
 * Pro C2
 * Helix terminaiton
 * Tyr HC2
 * Cross-links H and F helices

=**Effect of pH**=


 * Binding of O2 to Hb is dependent of pH
 * Lower pH, affinity of Hb to O2 was reduced
 * Higher pH, affinity of Hb to O2 was increased
 * His 146 is responsible for this effect
 * Sensitive to pH and can be protonated and deprotonated reversibly
 * Forms a salt bridge with Asp 94 when protonated
 * When not protonated, the salt bridge with Asp 94 is broken
 * Dramatic change in structure just from being protonated/deprotonated

=**Effect of CO2**=


 * CO2 binds to Hb-α-Val1-NH2
 * Froms carbamino hemoglobin
 * Carbanimo lysine on α subunit can form a salt bridge with Arge 141
 * Stablizes the T state
 * N-terminus of β subunit is also carbamylated
 * Allows effective transport of CO2

**CO2 Transportation**

 * Most CO2 is transported as HCO3-
 * Hydration/Dehydration of CO2 doesn’t happen fast enough so it requires an enzyme: carbonic anhydrase
 * Some is transported as HbCO2-
 * Very little is transported as CO2
 * pH at lung is higher than site of metabolism
 * At lung, favor binding of O2
 * At site of metabolism, favor disassociation of O2