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L- A partir de los datos de la tabla calcular la constante de disociacién de un ligando a una proteina: Tigando](mm) | ¥ OB 0,30 0:50 0,45 0,80 056 1,40 0,66 2,20 0,80 3,00 0,83 450 0,86 6,00 0,93 2+ éQué conjunto de datos podria indicar la union cooperativa de un ligando a una proteina? Tigandoal(mm) | ¥ Tigando ] (mm) | _¥ 02 or ol 03 03 03 02 Os om 06 Og 07 06 08 O7 09 3+ Algunos animales primitivos poseen una hemoglobina con dos subunidades idénticas. a) dibuja una curva de union de oxigeno para esta proteina. b) £ ual sera el rango de valores de su coeficiente de Hill? 4. En la hemoglobina Rai jer la Tyr 1456 esta sustituida por Cys que forma un enlace disulfuro con otra Cys de la misma subunidad previniendo la formacién de puentes salinos que estabilizan el estado T. £En qué se diferencia la hemoglobina Reinier con respecto a la normal en lotocante a: 1) afinidad por el oxigeno. 2) efecto Bohr. 3) coeficiente de Hill 5. Los buceadores a menudo se hiperventilan (respiran rapida y profundamente durante varios minutos) antes de hacer una inmersion en la creencia de que ello aumentara el contenido de oxigeno de la sangre. a) ZEs esto cierto?, b) Utiliza tus conocimientos sobre la hemoglobina para valorar si esta practica es realmente itil PRACTICAL STUDY CASE Focus concept A mutation in the gene for hemoglobin results in an altered protein responsible for the disease sickle cell anemia, An understanding of the biochemistry of the disease may suggest possible treatments. Prerequisite Hemoglobin structure and function concepts. Study case We will consider a patient, a 10-year-old black male child named Michael B., who was admitted to the hospital because he was experiencing severe chest pain. He had been hospitalized on several previous occasions for vvaso-occlusive episodes that caused him to experience severe pain that could not be managed with non- prescription drugs such as ibuprofen. He was slightly jaundiced, short of breath and easily tired, and feverish. A chest x-ray was taken and was abnormal. An arterial blood sample showed a p02 value of 6 kPa (normal is 10- 13 kPa). Questions 1. You suspect that Michael has sickle cell anemia and you have ordered an isoelectric focusing analysis of the child’s lysed red blood cells. (Lysing the red blood cells releases the hemoglobin.) Draw a diagram of the predicted results, Why will this test allow you to diagnose this child's disease? 2. Why do you think that Hb S molecules would be likely to clump together whereas Hb A molecules do not? 3. In the emergency room, oxygen (100%) was administered to the patient. (Inspired air normally is about 20% oxygen.) Why was this an effective treatment? 4. You recall reading in the medical literature about a new drug treatment for sickle cell anemia, and you'd like to try it on this patient. The drug is hydroxyurea, and is thought to function by stimulating the afflicted person’s synthesis of fetal hemoglobin. Exactly how hydroxyurea stimulated fetal hemoglobin synthesis is unclear, but it is believed that hydroxyurea is metabolized to NO, which binds to a soluble guanylate cyclase enzyme which then catalyzes the synthesis of a second messenger, cyclic GMP (cGMP). The cGMP interacts with transcription factors in a manner that is not completely understood to induce the transcription (and then translation) of the fetal hemoglobin gene. a. In a dinical study, patients who took hydroxyurea showed a 50% reduction in frequency of hospital admissions for severe pain, and there was also a decrease in the frequency of fever and abnormal chest x- rays. Why would increasing the synthesis of fetal hemoglobin result in alleviating the symptoms of sickle cell anemia? b. Medical practioners who used hydroxyurea as a treatment for sickle cell anemia noted that their patients seemed to benefit from the administration of the drug long before the synthesis of fetal hemoglobin had time to take effect. It has recently been determined that hydroxyurea can react directly with the iron ion of oxy- and deoxyHb to form iron nitrosyl hemoglobin (HNO). Why would this be of benefit to the sickle: cell anemic patient? And finally, hydroxyurea has been shown to produce NO directly, in less than an hour after hydroxyurea administration. It’s also possible that HbNO could produce NO, either directly or indirectly. NO is an important second messenger which, even in nanomolar amounts, stimulates vasodilation. How could NO production help the sickle-cell anemic patient? 5. Ayyear ago, at a conference, one of your colleagues told you that she had “cured” a patient of sickle cell anemia by performing a bone marrow transplant. Why would this procedure “cure” sickle cell anemia? PRACTICAL CASE: Allosteric Interactions in Crocodile Hemoglobin Focus concept ‘The effect of allosteric modulators on oxygen affinity for crocodile hemoglobin is unique when compared with other species. Background While most human beings are able to hold their breath for only a minute or two, other species are able to stay under water for much longer periods of time. In this case study we will examine the physiological adaptations that allow some organisms to deliver oxygen to tissues while submerged under water. Deep sea-diving mammals, such as whales and seals, are able to stay under water for long periods of time. These mammals are able to stay submerged because their muscles contain many-fold higher concentrations of myoglobin (Mb) than humans. Crocodiles are also able to stay submerged under water for periods of time exceeding one hour. This adaptation allows the crocodile to kill small mammals by drowning them. However, the crocodile doesn’t have large amounts of myoglobin in its muscle as the deep sea-diving mammals do, so their physiological adaptation must be different. In 1995, Nagai and colleagues described in the British journal Nature a possible mechanism that allowed the crocodile hemoglobin to deliver a large fraction of bound oxygen to the tissues. They suggested that bicarbonate, HCO3,, binds to hemoglobin to promote the dissociation of - ‘oxygen in a manner similar to 2,3-bisphosphoglycerate (BPG) in humans. Questions 1. In humans, oxygen is effectively delivered to the tissues because of the presence of several allosteric modulators. Name three of these modulators and explain how their presence allows oxygen to be delivered to thetissues, 2. Explain why having higher concentrations of Mb would allow whales and seals to stay submerged under water for a long period of time. 3. Let us consider the hypothesis that bicarbonate serves as an allosteric modulator of hemoglobin binding in crocodiles. What is the source of HCO3 in the crocodile tissues? 4. Draw oxygen-binding curves for crocodile hemoglobin in the presence and absence of bicarbonate. Which conditions give rise to a greater p50 value for crocodile hemoglobin? What does this tell you about the oxygen binding affinity for hemoglobin under those conditions? 5. _Komiyama et al. investigated the bicarbonate binding site on the crocodile hemoglobin by constructing human-crocodile chimeric hemoglobins in which amino acids in the human hemoglobin were replaced with amino acids found in the crocodile hemoglobin at the same location. (The investigators wanted to see if they could make a synthetic human hemoglobin that resembled the crocodile hemoglobin in terms of its ability to bind bicarbonate anions.) They found the bicarbonate binding site to be located at the a152-subunit interface, where the two subunits slide with respect to one another during R <> T transitions. Based on their results the authors modeled a stereochemically plausible binding site that included the phenolate anion of Tyr 418, the e-amino group of Lys 388, and the phenolate anion of Tyr 42a. What kinds of interactions do you think the aforementioned amino acid side chains will have with the bicarbonate anion? (It might be helpful to draw the Lewis electron dot structure of bicarbonate). 6. In order to create an engineered human hemoglobin molecule that had the same bicarbonate binding properties as crocodile hemoglobin, twelve amino acid residues had to be changed. Not all of these residues directly interact with bicarbonate-perhaps only three of them do, as described in question 5. What might be the role of the other nine amino acid residues? 7. Other animals have similarly adapted to using small molecules as allosteric effectors to encourage hemoglobin to release its oxygen. Whereas humans use 2,3-BPG and crocodiles use HCO3 , birds use myo-inositol pentaphosphate (IP5) and fish use ATP and GTP. What structural characteristics do all of these molecules have in common and how would they bind to hemoglobin? Reference Komiyama, N. H., Miyazaki, G., Tame, J., and Nagai, K. (1995) Nature 373, pp. 244-246.

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