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Lab9_experiment-1 - Topic 9 Experiment Ligand Binding to...

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Topic 9 Experiment Ligand Binding to Hemoglobin Introduction The chemistry of protein bound iron•heme complex is of keen interest since several heme proteins and enzymes (including catalase, peroxidase, cytochrome a 3 and cytochrome c) are involved in metabolic functions within the human body. The biological function of hemoglobin, like of many proteins and most enzymes, is maintained by highly optimized reaction dynamics and delicately balanced chemical equilibria. When iron is present in the +2 state, the ferrohemoglobin is capable of binding O 2 , and carrying out its biological function. This reaction can be written as hemoglobin•4Fe(II) (aq) + 4 O 2(aq) → Keq hemoglobin•4[Fe(II) – O 2 ] (aq) where hemoglobin•4Fe(II) represents ferrohemoglobin and the iron of the heme and hemoglobin•4[Fe(II) – O 2 ] represents the hemoglobin-oxygen adduct. The “4” of the “4Fe(II)” is a result of the fact that hemoglobin protein is composed of four subunits. Each subunit contains an iron•heme complex that can reversibly bind oxygen. In the lungs, oxygen from the air is bound by hemoglobin in the blood and then transported through the areteries to myoglobin in muscle cells. In order to successfully achieve this transport the oxygen binding constants of the hemoglobin to the oxygen must be the proper magnitude. The ability to measure equilibria is necessary if a detailed understanding of the biological function of proteins, such as hemoglobin, is to be obtained. Hemoglobin is one of several proteins and enzymes that relies on the chemical versatility and tenability of the iron ion. In these systems, the structural environment that defines the ligand field of the ion influences the chemical properties of the ion. These properties include the ease with which the iron is oxidized and reduced, the rate with which ligands (molecules) are exchanged, and the effective size of the iron ion. In this laboratory exercise, you will study the binding properties of the model hemoglobin-ligand system, ferrihemoglobin- imidazole. Fig. 10.3 shows the structure of imidazole, abbreviated Im . Through your laboratory study, you will determine the binding constant for the overall reaction. Analogous to myoglobin, ferrohemoglobin (Hb with 4 Fe 2+ ) can easily be oxidized to ferrihemoglobin (Hb + with 4 Fe 3+ ) using suitable oxidizing agents. When the iron is present in the +3 oxidation state the binding properties of the iron ion are modified; ferrihemoglobin binds to imidazole and ions, such as OH - and F - , rather than O 2 . Fig. 9.3 Imidazole structure
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Experimental Procedure To prepare samples for study by optical spectroscopy, first all solids must be removed from the samples. High speed centrifugation is an efficient way to remove suspended solids. Fill two 2-mL microcentrifuge tubes with equal amounts (~1.5mL each) of hemoglobin solution. It is very important that both centrifuge tubes contain equal volumes of solution. Place the samples across
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