Globular Protein .pdf

Globular Proteins (Hemoglobin/Myoglobin) 2024 OPT-517 UPIKE-KYCO B1 – 10 J. Michael Younger, Ph.D.  Objectives - Describe the general structure of Hemoglobin and Myoglobin - Compare and contrast the Oxygen Dissociation...

Globular Proteins (Hemoglobin/Myoglobin) 2024 OPT-517 UPIKE-KYCO B1 – 10 J. Michael Younger, Ph.D.  Objectives - Describe the general structure of Hemoglobin and Myoglobin - Compare and contrast the Oxygen Dissociation Curve for Hemoglobin and Myoglobin - Characterize the allosteric effectors associated with Hemoglobin function, along with the concept of Heme-Heme Interactions and Hemoglobin poisons - Discuss the utility of an A1c analysis and explain the increase in HbA1c - Characterize Hemoglobinopothies and the rational for their prevalence  Hemeproteins A group of specialized proteins that contain heme as a tightly bound prosthetic group Prosthetic group = coenzyme that is permanently associated with the enzyme (or other protein) and returns to its original form after substrate modification Examples of heme proteins: - Cytochromes: the heme group serves as an electron carrier that is alternatively oxidized and reduced. The main cytochromes discussed in human biochemistry are those of the electron transport chain (ETC). - Catalase: the heme is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide. This enzyme is found in the peroxisomes. - Myoglobin: heme is used to reversibly bind oxygen (Heart & Skeletal Muscle Cells) - Hemoglobin: heme is used to reversibly bind oxygen (Red Blood Cells)  Hemeproteins Structure of heme a) Heme is the most prevalent porphyrin in humans b) Iron (Fe2+) is held in the center of the heme molecule by four nitrogen residues of the porphyrin ring. c) The heme iron can form two additional bonds (six total) d) In Myoglobin and Hemoglobin: - one bond is with the R-group of a histidine residue of the globin protein - the other position is available to bind oxygen  Structure and Function of Myoglobin Myoglobin is present in heart and skeletal muscle: Increases 02 - serves as an oxygen reservoir & carrier Binding 02 Attuning - increases Oxygen Transport Rate within the Heart and Skeletal muscle cells - is a single polypeptide (similar too individual alpha and beta globin chains in tetrameric hemoglobin) - 80% of the molecule is composed of alpha-helix stretches that are flexible - the Heme sits in a crevice in the molecule lined with nonpolar amino acids except for two histidine amino acids - One histidine binds to the iron of heme, the other histidine is on a flexible alpha-helix that stabilizes the binding of oxygen to the iron (increased affinity)  Structure and Function of Hemoglobin Hemoglobin is found exclusively in red blood cells (RBCs) - its main function is to transport up to four O2 from the lungs to the capillary beds - it can also transport CO2 and H+ from the peripheral tissues back to the lungs. Hemoglobin A (HbA): is the major adult hemoglobin - composed of four polypeptide chains: two alpha-beta dimers (2 ab dimers) ab :: ab where: 1. the ab of a dimer are attached by non-polar or hydrophobic interactions 2. the ab :: ab dimers interact via reversable charge-charge and hydrogen bonding interactions Important to note: The structure of each of the four subunit is similar to the structure of myoglobin.  Structure and Function of Hemoglobin The Quaternary structure: the HbA tetramer can be envisioned as two identical dimers (alpha-beta)1 and (alpha-beta)2, and each alpha-beta dimer is held together primarily by hydrophobic interactions. The weaker ionic and hydrogen bonds hold the two alpha-beta dimers together to form the tetramer, and the amount/strength of polar bonding between the two dimers depends on the amount of oxygen that is bound  Structure and Function of Hemoglobin Heme Moravians Infdction T form (taut or tense): this is the deoxy form of hemoglobin when no oxygen is bound, and is also the low-oxygen-affinity form of hemoglobin 1 R form (relaxed form): binding of oxygen to hemoglobin causes a break in some of the polar bonds between the dimers, and this is the high oxygen affinity form of hemoglobin less Interactions T-Form High affinity R-Form 402 Affinity for O2  Structure and Function of Hemoglobin Heme-heme interactions – binding of oxygen to heme changes the structure of hemoglobin, such that it T-Form increases the affinity of the other heme groups for oxygen. Each oxygen bound increases the affinity, such that affinity for the last oxygen bound is approximately 300 times greater than its affinity for the first oxygen bound. R-Form Oxygen Histidine Heme a-helical of -globin  Structure and Function of Hemoglobin Heme-heme interactions affect loading and unloading of O2: Loading - at the lungs, the pO2 is high - hemoglobin will bind O2 - affinity for O2 increases due to O2 binding - nearly saturated (~100%) Unloading - at peripheral tissue capillary beds pO2 is low - O2 can disassociate from hemoglobin - affinity for O2 decreases due to O2 release - depending on oxygen demand at capillary bed and how low pO2 is, determines O2 disassociation Cooperative Binding and Disassociation allows hemoglobin to efficiently deliver appropriate amounts of O2 to the tissues in response to relatively small changes in the pO2. capillary pelias bed 8m'S  O2 Binding/Dissociation Curves Compare Myoglobin & Hemoglobin Y axis - degree of saturation (Y axis) X axis - Partial pressure of oxygen (pO2). Where P50 = partial pressure of oxygen (pO2) at which 50% of the O2 binding sites of myoglobin or hemoglobin are are saturated. This is similar to Km E - A low P50 = high affinity/left shift of curve (Heme needs a higher affinity for 50% 5090 loading/unloading at that pO2) rooms - A high P50 = low affinity/right shift of curve IT 60010 (Heme needs a higher affinity for 50% loading/unloading at that pO2) has to myo Globin high affinity 9 2 5 1 0 un's  capillary bed Portia pressure O2 Binding/Dissociation Curves vicers Compare Myoglobin & Hemoglobin 46 Myoglobin - can only bind one oxygen (O2) - hyperbolic curve - high affinity - narrow dissociation range Hemoglobin - can bind up to four oxygen (O2) - sigmoidal curve (suggests cooperative binding) - variable affinity- lower than myoglobin - Wider dissociation range Degree of saturation (Y axis) of the oxygen binding sites on all myoglobin or hemoglobin molecules can vary between 0% (all sites are empty) and 100% (all sites are full). % Saturation vs P50  O2 Binding/Dissociation Curves Compare Myoglobin & Hemoglobin Myogobin: hyperbolic shape (like simple enzymes), very low P50 (very high oxygen affinity), much higher than hemoglobin. Myoglobin is designed to bind oxygen at the low pO2 found in muscle, and releases oxygen in response to oxygen demand. Hemoglobin: sigmoidal shape indicating cooperative binding where Heme-heme interaction = any change in affinity for oxygen at one site affects the affinity for oxygen at the other three sites - binding of one oxygen molecule at one site increases the affinity for oxygen at all sites  O2 Binding/Dissociation Curves Significance of the sigmoidal oxygen dissociation curve: - the steep slope of the oxygen dissociation curve over the range of oxygen concentrations that occur in the peripheral tissues allows hemoglobin to carry and deliver appropriate amounts of oxygen from sites of high to low pO2. that Tetramer bind to 02 can Intracdal Heme Heme parola pressure that Factor stagination drives T Form Of  more Acid CADET Face Decrase in In o O2 Binding/Dissociation Curves pusha or Allosteric Effects (Hemoglobin) Interaction of allosteric effectors at one site on the hemoglobin molecule affects the binding of oxygen to heme groups at other locations on the molecule Allosteric Effectors: -- pCO2 (CO2 binding) -- Bohr Effect (pH) (ie: H+ binding) -- 2,3-BPG (2,3-BPG interaction between ab Dimers) Each of these affect the P50 and cause an apparent shift in the O2 Dissociation Curve for Hemoglobin to accommodate the O2 needs of tissues Note: all allosteric effectors decrease Hemoglobin affinity for O2 and shift the curve to the right, which increases O2 dissociation at a particular pO2. All allosteric effectors promote off-loading. (more accurately, they stabilize the low affinity T-Form) Allosteric Effectors - pCO2 Binding of CO2 some CO2 is transported as carbamate bound to the N-terminal amino groups of hemoglobin forming carbaminohemoglobin: Hb-NH2 + CO2 ß à Hb-NH-COO- + H+ COO- binding stabilizes the T-form of hemoglobin Lower affinity T-form causes apparent right shift in the oxygen dissociation curve at the capillary bed and increased off-loading of O2 At the lungs, CO2 dissociates from the hemoglobin and is released in the breath Note: Under less demanding conditions, T-form is less likely and most CO2 is transported in the form of bicarbonate ion.  Allosteric Effectors - Bohr effect (pH) Bohr effect (pH): Hemoglobin has less affinity for oxygen at lower pH which causes an apparent right shift in the oxygen dissociation curve at the capillary bed and increased off-loading of O2 Source of protons to lower the pH: Carbaminohemoglobin formation: Hb-NH2 + CO2 ß à Hb-NH-COO- + H+ Organic acids produced by tissues, such as lactic acid from rapidly contracting muscle CO2 at peripheral tissues results in production of carbonic acid, carbonic anhydrase and bicarbonate (CO2 + H2O à H2CO3 à HCO3- + H+ ) Because of this effect of CO2, there is a pH gradient between the lungs and peripheral tissues, where the pH is higher in the lungs and lower in the peripheral tissues, increasing the efficiency of hemoglobin as an oxygen transporter.  Allosteric Effectors - Bohr effect (pH) Bohr effect (pH): Mechanism of the Bohr effect: - Deoxyhemoglobin (T-form) has a higher affinity for protons than oxyhemoglobin Explanation: - histidine R-groups have a higher pKa when not bound to O2 - become protonated - Histidine R-group will have a (+) Charge - protonated deoxyhemoglobin forms stronger ionic interactions between ab dimers - more stable T-form - lower affinity for oxygen - doesn’t re-bind O2 at capillary bed  Allosteric Effectors - 2,3-BPG REG 10955 Effect of 2,3-bisphosphoglycerate (2,3-BPG) on oxygen affinity: 2,3-BPG is only found in significant concentration in RBCs, it is a side product of glycolysis and can be isolated from metabolism by Hemoglobin. Binding of 2,3-BPG to deoxyhemoglobin: – 2,3-BPG binds to deoxyhemoglobin, but not oxyhemoglobin – stabilizes T-form, Forms – therefore decreases the affinity of hemoglobin for oxygen T Fom active physically tlaborg more to 213 BPGhemoglobin deoxy Allosteric Effectors - 2,3-BPG Effect of 2,3-BPG - function in in Binding site: Binds in pocket between two beta-globin chains in deoxyhemoglobin - which contain positively charged amino acids that bind to the negatively charged 2,3-BPG - stabilizing the T-form. - When oxygen is bound, 2,3-BPG is expelled - The net effect of 2,3-BPG results in a shift to the right of the oxygen dissociation curve, - which puts the steepest part of the curve in the range of the pO2 found at the peripheral tissues.  Allosteric Effectors - 2,3-BPG Effect of 2,3-BPG – compensation with hypoxia & anemia In conditions such as chronic hypoxia or anemia – 2,3-BPG Level increases to compensate for O2 deficiency hypoxia -- 2,3-BPG levels rise in chronic hypoxia COPD – chronic obstructive pulmonary disease or high altitudes result in poor O2 loading at the lung and subsequent deficiency at capillary bed. - poor O2 exchange in COPD - lower atmospheric pO2 at high altitudes -- In anemic patients, with less hemoglobin and/or RBCs, 2,3-BPG levels are also increased to allow more efficient unloading of oxygen to peripheral tissues m more 11kg to get 2 3 BPG levels  Allosteric Effectors - 2,3-BPG Effect of 2,3-BPG – blood transfusion consideration - Role of 2,3-BPG in transfused blood: -- 2,3-BPG is essential for normal transport function of oxygen by hemoglobin -- without it the oxygen dissociation curve shifts to the left and has affinity that is too high -- Hemoglobin binds oxygen with such high affinity, it can’t unload oxygen to peripheral tissues efficiently. -- During storage of blood, 2,3-BPG is metabolized, large quantities of this blood can compromise patients because it will bind oxygen too tightly until 2,3-BPG levels return to normal (6-24 hours). Toxic Effect of CO (Carbon monoxide poisoning) Binding of CO: Hemoglobin has 200x the affinity for carbon monoxide as it does for oxygen, and therefore binds it very tightly (with High Affinity). It causes hemoglobin to shift to the relaxed form and bind oxygen with very-high affinity, This shifts the apparent oxygen dissociation curve to the left. a combination of high oxygen affinity and oxygen binding sites occupied by CO results in critically low oxygen unloading at peripheral tissues. 6m to 1 or Drug hemostoon for affinity oxygen Attining and up Yous up  know this slide Minor Hemoglobins 1. Fetal hemoglobin (HbF) APha Gamma two alpha chains and two gamma chains (ag::ag) The gamma-chains lack some of the positively charged amino acids found in the beta-chains that interact with 2,3-BPG, curve shifts to 1171g and therefore has lower affinity for 2,3-BPG. 02 This results in HbF having a higher affinity for oxygen, Increased affinity of HbF allows oxygen extraction from maternal circulation across the placenta.  Minor Hemoglobins A 2. Hemoglobin A2 (HbA2): Au't Herosion - minor hemoglobin (2%) - unknown function  Minor Hemoglobins 3. Hemoglobin A1c (HbA1c): HbA is slowly and non- concurrently Glucose enzymically glycolated under physiological conditions, forming HbA1c. The extent of this glycolation is dependent on the concentration of glucose that the RBC is exposed to Hr All over its 120-day lifespan. Mandolny Higher concentrations of blood glucose, such as glucose those seen in patients with diabetes mellitus, will 6 081 look result in higher concentrations of HbA1c. levers The A1c test is particularly valuable in testing for the on-set of diabetes and the long-term efficacy of a treatment plan, or monitoring a patient’s adherence to a treatment plan to control blood glucose  All more Hemoglobinopathies access 1. Sickle cell anemia (hemoglobin S disease): a) Most common inherited blood disorder b) Like beta-thalssemia, symptoms are not seen until around ~6 months of age (when HbF is replaced by HbS). c) Symptoms: episodes of pain (crises), chronic hemolytic anemia (and hyperbilirubinemia), increased susceptibility to infection d) Lifetime of RBC = less than 20 days e) Mutation = glutamate replaced by valine, less negatively charged, migrates slower under electrophoresis GluàValàLys (-) (0) (+) happening Hpa is 4pm has mutation the beta in chain mutation is due to parasite  Invertan 1. Sickle cell anemia (hemoglobin S disease): f) Sickling and tissue anoxia: The replacement of a glutamate with a valine creates a protrusion on the beta-globin that fits into a complementary site on a beta-globin on another hemoglobin molecule in the RBC. At low oxygen tension (low pO2 T-form of hemoglobin) the hemoglobin polymerizes forming a network of fibrous polymers that stiffen and distort the cell. Sickled cells can lodge and interrupt the flow of blood in capillaries, causing anoxia (oxygen deprivation) in the tissue that causes pain and eventually death (infarction) of the tissue. 2 a Has snet crus 1. Sickle cell anemia (hemoglobin S disease): g) Variables that increase sickling: Disease severity (sickling) is increased by any variable that increase the proportion of the hemoglobin in the deoxy state: ie, high altitudes, flying in a nonpressurized plane, increased CO2, decreased pH, dehydration, increased concentration of 2,3-BPG. 1. Sickle cell anemia (hemoglobin S disease): h) Treatment: hydration, analgesics, antibiotic therapy, blood transfusions (must weigh benefit vs. risk). Hydroxyurea (antitumor drug) can increase the amount of HbF, which decreases RBC sickling. Fromm increases Hbf Note: The cause and organism responsible for the problem: -- parasitic Plasmodium infection -- 1. Sickle cell anemia (hemoglobin S disease): A. Sickle cell distribution expressed as a percentage of the population with the disease B. Distribution of malaria in Africa  Homozygous Disease condition Hemoglobinopathies core gen 2. Hemoglobin C disease : lysine substituted for glutamate (same position as mutation for HbS), homozygotes have relatively mild chronic hemolytic anemia, no infarctive crises, IAIN no specific therapy required. mutating 3. Hemoglobin SC disease: when a patient inherits one copy of HbS and one copy of HbC. Symptoms are variable and somewhere between the more severe sickle cell anemia, and the less severe Hemoglobin C disease. They do get some sickling and resulting crises, but are less frequent and less severe. 12 alleles one of union Jonas exorasses 1 HBS e me HBC one gets crisis 2 seeing taxemns