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Chapter 7: Student Notes for Objectives Objective Shortcut: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18 19 20 21 22 23 24 25 26 27Objectives, Key Words, Assignments, Practice Questions, Other Help,

Home Page. Objectives: 1. Understand the meaning of primary, secondary, tertiary, and quaternary structure and be able to discern the difference. Primary Structure: Is linear sequence of amino acids in polypeptide chain. e.g. ala-gly-leu-arg-leu-, etc. Written from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). Determines the way a protein folds into a unique three-dimensional conformation (native conformation). Secondary Structure: 1. Alpha helix, 2. beta-pleated sheets, and 3. bends, turns, or loops (nonrepetitive secondary structures). Localized (short) areas of the polypeptide chain form these structures that are stabilized by hydrogen bonds Alpha helices are formed when carbonyl group of peptide bond forms a hydrogen bond with the amide nitrogen of another peptide bond four amino acids down the polypeptide chain. Beta-sheets are formed when beta strands are connected laterally by at least two or three backbone hydrogen bonds. Tertiary Structure: Tertiary structure is the total 3-D conformation of an entire polypeptide chain including: alpha-helices, beta-sheets and any other loops, turns, or bends. Every protein has 1 or 2 most stable conformations. Quaternary Structure: A combination of two or more tertiary subunits that work together as one functioning unit. Hint: It helped me to visualize the differences between the structures by using one of those coiled telephone cords. If you straighten it out so it no longer coils, it is in the primary structure. If you let it relax back into the coils, it is in the secondary (alpha helix) structure. If you ball the whole thing up, it is now a tertiary structure. If you take several balled up, coiled telephone cords and put them together, it is a quaternary structure. they would have 1 or 2 most stable conformations.

Top 2. Be able to recognize and discern between the three types of secondary structure discussed in class! Alpha Helix:

The peptide backbone is formed by hydrogen bonds between each carbonyl oxygen atom and the amide hydrogen located 4 residues down the chain. This unique bonding sequence results in an alpha helix struture that is highly compact and rigid. Comprises about a third of all secondary structures. R-groups protrude from the helix See Figures 7.3 and 7.4 Beta Pleated Sheets: Are made up of Beta strands that are almost fully extended in the sheet. Two or more beta strands form a beta sheet. The beta strands are connected laterally by hydrogen bonds formed between C=O groups of either strand and NH groups of either strand. When the sheet is pleated optimal hydrogen bonding occurs. The pleats are in the plane formed by the beta strands. Beta pleated sheets can either be parallel or antiparallel. If the amino terminals and the carboxyl terminals are all on one end, then it is parallel. In antiparallel, amino and carboxyl terminals are on opposite ends. The R groups always protrude to the top or bottom. See Figure 7.5. Non-Repetitive Structures: Bends, loops, and turns. Are best recognizable as forms that resemble neither an alpha helix nor a beta pleated sheet. These do not have a repeating element in their amino acid sequence. These structures are often found on the surface of the protein. See Figure 7.6.

Top 3. What is the location of the R-groups in an alpha helix? Can an alpha helix be formed from any primary sequence of amino acids? R groups project outward, away from the central axis of the helix. See figure 7.4. No, only certain sequences of amino acids can form an alpha helix. Runs of positively or negatively charged R-groups can disrupt helix because of electrostatic repulsion. Runs of bulky R-groups can disrupt the helix with their steric hindrance. Proline breaks the helix formation and therefore can not be in a helix because it can't form the correct bond angles.

Top 4. What is the difference between parallel and antiparallel -sheets? How do the R-groups protrude from the sheets? Beta sheets are parallel if the polypeptide strands run in the same direction, N-terminus to C-terminus. The N-terminus of one beta strand will be opposite the N-terminus of the other beta strand.

Beta sheets are anti-parallel if the polypeptide strands run in opposite directions. The N-terminus of one beta strand will be opposite the C-terminus of the other beta strand. R groups protrude from the top and bottom of the beta sheet. The R groups on one side of a beta-sheet tend to be either all hydrophilic or all hydrophobic. The hydrophilic side of the sheet will be on the surface of the protein and be exposed to the polar H2O solvent. The hydrophobic side of the sheet will be buried in the protein and will only rarely be exposed to the polar H2O solvent.

Top 5. What are the three names for nonregular, nonrepetitive secondary structures? Bends, Loops, and Turns Bends, loops and turns are secondary structures that don't repeat like the alpha helix or beta pleated sheets. That is, if you looked at the bends, loops, and turns of ten proteins, the structure of the backebone of the bend, loop or turns would be different in each. They are often found on the protein surface.

Top 6. What is a motif? A motif is an arrangement of a small number of secondary structures recurring in many different proteins. A motif is a level of protein structure between secondary structure and tertiary structure. A motif is a short recurrent segment of a proteins three-dimensional structure that is found in a large number of different proteins. A motif may contain 2 to perhaps 7 or 8 elements of secondary structures. Example: the beta-alpha-beta motif consists of three secondary structures; a beta strand connected to an alpha helix connected to another beta strand. Another example would be: beta-alpha-loop-alpha-turn-beta. Motifs cannot exist on their own. They require the rest of the domain to maintain their conformation.

Top 7. What is a structural domain? Structural domains are physically independent regions of the tertiary structure that can maintain their conformation without the rest of the protein. Structural domains have a specific function (perform a particular physical or chemical task). often fold by themselves from the primary structure.

may function alone or combine with other domains to form a functional tertiary structure. contain alpha-helicies and/or beta-sheets in addition to a number of loops, turns or bends. Proteins are found to have domains in common even if their overall tertiary structures and functions are different. Often the term domain is used loosely by biologists to describe a portion of a protein that catalyzes a reaction or binds to something. This is so common that it is the norm.

Top 8. What is a fold (fold structure or protein fold)? What is a fold family? See Chapter 7, Other Help Folding is the process by which a primary and secondary structure folds into its tertiary conformation. A Fold (fold structure or protein fold) is simply a tertiary structure that has resulted from the folding of the primary and secondary structures. However, when talking about a fold instead of the tertiary structure we are emphasizing and examiningeach segment of secondary structure and how it is related in space to every other segment of secondary structure. That is, we are looking at how the alpha-helices, beta-strands, loops, turns, and bends folded relative to each other in space. Proteins are defined as having a common fold if they have the same major secondary structures in the same arrangement and with the same topological connections. This does not mean they are related by evolution. A Fold Family contains proteins that have the same major secondary structures in the same arrangement and with the same topological connections and that areclearly related by evolution. Other Information: A Superfamily contains proteins that have the same major secondary structures in the same arrangement and with the same topological connections but whether they are related by evolution is not clear. It is believed there have only been a few thousand folds for all the proteins that ever existed. Folds are formed because of the thermodynamic stability.

Top 9. When we say that actin, heat shock protein 70, and hexokinase are homologous, what are we implying? Protein homology means that the proteins are derived from a common ancestor. Duplication of the gene followed by mutations has produced two similar proteins that may even have different functions. Because they are evolved from the same ancestor gene, the homologous proteins will have the same number and types of secondary structures oriented in the same way in space.

When we say that actin, heat shock protein 70, and hexokinase are homologous, we are referring to the actin fold that is a part of each of these proteins. The actin fold of each of these proteins is clearly in the same fold family, i.e., is clearly related by evolution. The rest of these proteins may be different, but the actin fold is in the same fold family. The three dimensional drawings of the actin folds of all three proteins are all superimposable. That is, they have the same major secondary structures in the same arrangement in space relative to one another. In all of these proteins, ATP binding results in large conformational changes that contribute to the function of the protein.

Top 10. Where would you most likely find the nonpolar, polar and charged amino acids in a globular protein? Nonpolar amino acids are most likely found in the hydrophobic interior of proteins (Val, Leu, Ile, Met, and Phe). Polar amino acids that are charged and polar amino acids that are not charged are most likely found on the hydrophylic surface (exterior) of proteins. (Charged: Arg, His, Lys, Asp and Glu. Polar: Ser, Thr, Asn, Gln, Tyr and Trp)

Top 11. In the beta-adrenergic receptor, where do you find the bound carbohydrate, the ligand binding site, the 7 alphahelical domains, and the G-protein binding site? Why is part of the polypeptide chain in the membrane? When the hormone binds, does the receptor change its conformation? In a transmembrane protein, the carbohydrate is usually found attached to the part of the protein that is outside of the cell. Since the hormone binds to the outside of the cell, the ligand binding site is outside of the cell. The 7 alpha-helical domains with their hydrophobic R-groups are in the hydrophobic environment of the membrane. G-Proteins are inside of the cell so the G-protein binding site of the receptor has to be inside of the cell. Part of the polypeptide chain is in the membrane because it is hydrophobic. When the hormone binds to the receptor, it forces a change in conformation of the receptor inside of the cell.

Top 12. What are the forces that hold the monomer units of a quaternary structure together? Hydrophobic interactions, hydrogen bonding, ionic bonds (salt bridges), and disulfide bonds. Except for disulfide bonds, these are the same forces that are responsible for the tertiary structure. Permanent disulfide bonds form after the formation of tertiary structures so they don't determine the conformation of the tertiary structure. However, disulfide bonds do help to stabilize the tertiary conformation.

Disulfide bonds are critical in holding the quaternary structure together. For example, if you hydrolyze the disulfide bonds in an immunoglobulin, the chains will drift apart and will never reform.

Top 13. For a ligand-protein complex that dissociates into a ligand and protein. Be able to state the dissociation constant and the association constant. See Equation 7.1 in textbook. LP __> L+P, This is the dissociation reaction L+P __> LP, This is the association reaction Kd = ([L][P])/ [LP] = 1/Ka Ka = [LP]/([L][P]) = 1/Kd [LP] is the concentration of the ligand-protein complex. [L] is the concentration of the ligand [P] is the concentrations of the protein. In practical terms, the stronger the bond between the ligand and the protein, the higher the Ka will be, and the lower the Kd will be. Ka is useful for describing the affinity a particular drug has for the protein it affects.

Top 14. Using the terms ferrous iron, heme, hydrophobic pocket, histidine, alpha-helix, alpha-turns, salt bonds, hydrophobic interactions, hydrogen bonds, oxygen, and subunits, describe the myoglobin and hemoglobin molecule. Myoglobin and Hemoglobin (Hb): Myoglobin and Hemoglobin are both oxygen binding proteins. Hemoglobin travels in the blood inside a red blood cell to deliver oxygen to tissues. Myoglobin remains in the heart and skeletal muscle cells to bind oxygen released by hemoglobin. The single globin chain of myoglobin and the four globin chains that make up hemoglobin are all homologous proteins and each of the polypeptide chains folds into a similar tertiary structure. Like all proteins, the tertiary and quaternary structures are stabilized byhydrophobic interaction, hydrogen bonds, and salt bonds (no disulfide bonds in these proteins). Myoglobin: -is a monomer has 8 alpha-helices linked together by alpha-turns. (No beta sheets/strands). -has a hydrophobic pocket containing heme with an ferrous iron atom (Fe2) at its center. This is the oxygen binding site. -Fe2+ is always bound to a histidine R-group of the alpha helix. This binding stabilizes the plus 2 state of iron when it binds to oxygen, i.e., keeps it from being oxidized to Fe3+ -heme is tightly bound to the globin so it is a prosthetic group. -consists of a single polypeptide chain that is homologous to the alpha- and beta-subunits of hemoglobin.

Hemoglobin (Hb): -Hb is a heterotetramer (four "mers" and at least one monomer is different from the others). -Hb is composed of 2 alpha and 2 beta subunits. -Each subunit has its own heme and, therefore, its own O2 binding site. -When O2 binds to the Fe2+ at one of the Hb binding sites, it pulls on the histidine, which pulls on the alpha helix, changing the conformation of the globin slightly. This slight movement changes the conformation of the other three chains in the Hb. This changes the oxygen binding affinity of the other oxygen binding sites and results in cooperativity -Cooperativity is positive, i.e., when oxygen binds to one heme, the other hemes are more likely to bind a second molecule of oxygen. When two oxygens are bound, the remaining two are more likely to bind oxygen than before. When three oxygens are bound, the remaining site is more likely to bind oxygen than before.

Top 15. Be able to draw an oxygen saturation curve for hemoglobin and myoglobin. See Figure 7.11, The curve shows the percent saturation with oxygen vs. oxygen pressure (concentration). The myoglobin curve is a rectangular hyperbola but the hemoglobin curve is an S-shaped (sigmoidal-shaped) curve because of cooperativity. Hemoglobin does not bind oxygen as strongly as myoglobin. However hemoglobin binds oxygen strong enough so that it is almost saturated with oxygen in the lungs where the partial pressure of oxygen is about 90 - 100 mm Hg. Hemoglobin releases oxygen in the tissues where the partial pressure of oxygen is about 20 - 40 mm Hg. When oxygen is released from hemoglobin, the loss of one molecule of oxygen facilitates the loss of additional oxygen molecules, This is the reverse of positive cooperativity'''. If hemoglobin was bound to oxygen as strongly as myoglobin is bound to oxygen, hemoglobin would never release oxygen when it reached the tissues.

Top 16. What is a ligand? What is an apoprotein? What is a holoprotein? What would an apoenzyme be? Ligand: Anything that binds to a protein. -Oxygen, heme, cofactors, coenzymes, metal ions, substrates, hormones, or even other proteins are examples of ligands. Apoprotein: A protein missing its ligand or ligands. Usually not functional without the ligand. Holoprotein: A protein with its ligand so it is able to function. Apoenzyme: An enzyme missing its cofactor or cofactors. When combined with the proper cofactors, usually a metal ion or a coenzyme, the apoprotein becomes an active enzyme (holoenzyme).

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17. What is a prosthetic group? A prosthetic group is a tightly bound structure required for the activity of an enzyme or other protein. For example, the tightly held heme of hemoglobin. Another example is a cofactor that is tightly bound. Cofactors are metal ions or coenzymes.

Top 18. Be able to explain why the oxygen saturation curve for hemoglobin is sigmoidal while the curve for myoglobin is a rectangular hyperbola. Include the terms subunits, O2, Fe2+, conformation, salt bridges, T-state, R-state. Reference Figure 7.11 for oxygen saturation curve and 7.14 for T and R states. Hyperbolic Curve: Myoglobin is a single polypeptide chain so the conformational change that occurs when O2 binds reversibly to Fe2+ has no effect on the conformation or O2 binding on other myoglobin molecules. Since there is not interaction between the myoglobin molecules, there is no T-state or R-state. As oxygen concentration is increased from zero, a rectangular hyperbola results. At low concentrations (pressures) of O2 a large amount of the added oxygen binds to the myoglobin molecule because there are plenty of binding sites available and the rate of dissociation of bound sites is low. For each increment of added oxygen pressure, a smaller increment of oxygen will be bound because there are fewer available binding sites and the rate of dissociation from bound sites will increase. The closer one gets to 100% saturation, the more difficult it is to bind more oxygen. Sigmoidal Curve: The sigmoidal curve results from the cooperativity of O2 binding in hemoglobin (Hb). Hemoglobin is a tetramer consisting of four polypeptide subunits (chains) that binds oxygen reversibly. Each of the monomers is a globin chain that looks and acts much like myoglobin with one big difference. The globin chains are associated with each other with salt bonds that decrease their affinity for oxygen. When the salt bonds are broken, the new conformation has a higher affinity for oxygen. Hemoglobin can exist in two conformations: the T-state or the R-state. In the T (tense) state, Hb has a low affinity for O2. In the R (relaxed) state, Hb has a high affinity for O2. The higher the oxygen pressure, the higher the proportion of hemoglobin molecules in the R-state. At lower levels of pO2 (the T-state) Hb cannot bind O2 as well as myoglobin, therefore its % saturation is much lower. If no O2 was present, most of the Hb would be in the T-state but some would be in the R-state and able to bind to O2. If O2 is added, it would bind to the Hb in the R-state and stabilize the R-state. The R-state will not go back to the T-state as long as one heme is bound to O2 so the proportion of Hb in the R-state would increase. Also, the other three oxygen binding sites on the Hb would have an increased affinity for O2. It follows that as the oxygen pressure is increased, the affinity of Hb for O2 would increase.

That is, as O2 concentration is increased in increments, starting from zero, each increment of O2 increases the incremental amount of bound O2 until the hemoglobin solution is half saturated. After the hemoglobin is half saturated with O2, a smaller increment of O2 will be bound because there are fewer available binding sites and the rate of dissociation from bound sites will increase. The closer one gets to 100% saturation, the more difficult it is to bind more O2.

Top 19. Be able to describe the structure of IgG in terms of the number of chains, the number and types of domains, and the forces that hold the chains together. Which part binds to an antigen and why is it specific? What fold family do the domains belong to. Reference Figure 7.15 The IgG immunoglobulin (antibody) consists of 2 smaller polypeptide chains (light chains) and 2 larger polypeptide chains (heavy chains). Each light chain is bound to a heavy chain by disulfide bonds and the heavy chains are bound to each other by disulfide bonds. Each heavy chain has 4 domains and each light chain has 2 domains so the IgG has a total of 12 domains. There are two types of domains: Variable Domains: variable regions for antigen binding Constant Domains: the same for all immunoglobins Each light and heavy chain has on variable domain and the rest are constant domains. There are millions of different variable regions found in a person, each one specific for one or a few antigens. The variability is due to many processes, including DNA recombination. Each variable domain is a beta-barrels with loops that stick out and bind to antigens. They bind to specific antigens because they have different conformations with different charge distributions. All the domains, both variable and constant, are collapsed beta-barrel made from multiple beta-sheets. They all belong to the immunoglobulin fold family. This means that they all evolved from a common gene. The forces that hold the chains together in immunoglobulins are hydrophobic interactions, hydrogen bonding, ionic bonds (salt bridges), and disulfide bonds.If one hydrolyzes the disulfide bonds, the chains drift apart and normal function is lost.

Top 20. What is the general name for the class of proteins that help other proteins fold into their native tertiary conformation? Heat shock proteins

Top 21. What is denaturation? Name a few ways that proteins are denatured in humans. Denaturation is a process in which the folding structure of a protein is altered due to exposure to certain chemical or physical factors causing the protein to become biologically inactive. The physical forces and chemical reactions disrupt ionic, hydrogen, hydrophobic bonds, and disulfide bonds maintaining the native conformation. Examples: Nonenzymatic glycosylation: glucose binds to an exposed amino group on a protein to form a denatured glycosylated protein. This mechanism of denaturation is thought to be important in the pathology of diabetes. Acid denaturation (pH denaturation): HCl in gastric juice interferes with the protein's hydrogen and ionic bonds and disrupts the native conformation to make the protein a better substrate for digestive enzymes. Heat denaturation: Increasing the temperature will increase vibrational and rotational energies in the bonds and break them apart (ex. cooking an egg: albumin converts from its native translucent state to a denatured white precipitate). Cold temperatures can also denature some proteins. Enzymes Denaturation: Enzymes unfold and lose their active state when exposed to denaturing agents (e.g. strong acids or bases, heat, solvents, and salts). When enzymes lose their structure, they lose their function as catalysts, i.e., substrates can no longer bind to the active site. If enough denaturation takes place, the enzyme reaction and the pathway it is part of fails. Other: Denaturation usually results in the permanent loss of protein function. However, under certain conditions, some denatured protein can refold into their native conformations and regain their original functions. This is called renaturation. For example, ribonuclease is denatured with organic solvents such as urea that disrupt hydrogen-bonding patterns and convert the protein to a soluble random coil that has no activity. If the denaturing agent (urea) is removed,, ribonuclease, which is a simple single-subunit protein, can refold spontaneously into its native conformation. Even some complex multisubunit proteins containing bound cofactors can sometimes renature spontaneously under the right conditions.

Top 22. What is a prion?

A prion (proteinaceous infectious agent) is a protein that causes slow but progressive brain degeneration in a host organism resulting in death. Top

23. Explain how a prion can cause dementia and death. Include the terms PrPc, PrPsc, template, activation energy, cascade, amyloid protein, and proteolytic degradation. Definition of Terms: PrPc is the normal conformation of the protein. PrPSc is the disease-causing conformation. It is autocatalytic and resistant to proteolysis. Although PrPSc and PrPc have the same amino acid composition, the PrPsc conformer is substantially enriched in B-sheet structure compared with the PrPc conformer, which has little or no B-sheet structure and is about 40% alpha-helix. The Sc stands for scrapies, the name given to the disease when it occurs in sheep. Template ,as in molecular template, is a molecule (as of DNA) that serves as a pattern for the generation of another macromolecule. Activation energy is the energy that must be put into a chemical reaction in order for the chemical reaction to occur. The higher the activation energy, the less likely the reaction is to occur.(see figure 8.7) Cascade: a series of chemical reactions that occur a result of a single trigger reaction or compound. Amyloid proteins are insoluble fibrous protein aggregates found in amyloid plaques. In this case, a large number of molecules of PrPsc , interact and precipitate together to form amyloid plaques. The name amyloid comes from the early mistaken identification of the substance as starch. Proteolytic degradation: the conversion of proteins into amino acids. Explanation: Most cases of Priion disease are sporadic, which means they occur in people without any known risk factors or gene mutations so that will be the type explained here. Somehow a molecule of PrPSc is made from PrPc to begin the disease. PrPSc contains no nucleic acids; therefore, it cannot self-replicate. Rather,PrPSc acts as a template to misfold normal cellular proteins PrPc intoPrPSc, a form that cannot undergo proteolytic degradation. The template lowers the activation energy barrier for the conformational change of PrPcto PrPSc, a reaction that would otherwise never take place. The refolding of PrPc to PrPSc initiates a cascade as each new PrPSc formed acts as a template for the refolding of additional PrPc molecules to disease-causing ones. Amplification of the reaction increases with time so that a single PrPSc ends up making millions and then billions of itself. As the concentration of PrPSc increases in the cell, they aggregate and precipitate outside of the cell to form amyloid plaques that are resistant to proteolytic degradation. So as the cascade continues, a stringy buildup of amyloid protein plaque results outside of the cell. As a result, the brain starts to shrink, causing a progressive disruption of normal brain function. This decrease in the number of neurons leads to dementia and eventually death. Other Information: Other human prion disease are Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, and kuru. They are all very rare. Creutzfeldt-Jakob disease is a familial forms of prion disease. It is inherited in an autosomal dominant pattern or is the result of a new mutation.

Variant Creutzfeldt-Jakob disease results from consuming contamined beef (cows withMad Cow Disease). This form of prion disease is not inherited. Kuru is a form of prion disease transmitted among members of the Fore tribe of Papua New Guinea via cannibalism. After death, the members of the tribe ate the brains of their recently departed colleague. This form of prion disease is not inherited. Mad Cow Disease is transmitted through the ingestion of nervous tissues or bone marrow of infected ovine or bovine. The term "Mad" in Mad Cow Disease in a reference to the way the animal is acting (crazy) not the emotion (anger).

Top 24. Concerning Will Sichel: In biochemical terms, explain the development of pain during a sickle cell crises. Include the specific mutation, oxygen pressure, and protein conformation in your answer. Will has sickle cell disease that is the result of a point mutation (missense mutation) at the 6th residue of the betaglobulin chains of hemoglobin. The mutation results in the substitution of a valine (neutral charge, hydrophobic) for glutamate (negative, hydrophilic). This mutation results in a change inconformation of the hemoglobin molecule. A hydrophobic area appears on the surface of the heme molecule when it is not bound to oxygen. The hydrophobic areas of many hemoglobin molecules stick together to form polymers (see figure in margin of text). The HbS polymers deform the normal biconcave disc shaped Red Blood Cell into a sickle shaped (crescent-moon) cell that is not as easy to deform as a regular cell. Red cells usually deform as they pass through the capillaries. The sickle cells tend to clump up and clog the capillaries. When When the patients blood oxygen pressure is low, as it is during vigorous exercise, polymerization is more likely. Also, the capillaries always have the lowestoxygen pressure so clumping is more likely in the capillaries. The clumping in the capillaries stops oxygen from reaching the tissue serviced by the capillary. The pain part is not explained except that where there is a lack of oxygen, there is usually pain.

Top 25. Concerning Anne Jeina: What are the three different proteins that appear in the blood following the beginning of a myocardial infarction? What are their normal cell functions? How soon can they be detected? How specific are they? Myoglobin: Normal cell functions? Binds O2 released by hemoglobin and stores it to meet the demands of muscle contraction. During contraction myoglobin releases the O2 which is picked up by cytochrome oxidase, an enzyme in the electron transport chain with a higher affinity for oxygen than myoglobin. How soon can it be detected? Within 2 to 4 hours following onset. So, fastest of the three. How specific is it for diagnosis?

It is non-specific for cardiac injury because it could also be an indicator of any type of skeletal muscle injury. Has a very high negative predictive value within 2-6 hours after the onset of the symptoms, i.e., if myoglobin is not present, then no muscle tissue has been damaged. CK (Creatine Kinase): Normal cell functions? Creatine Kinase catalyzes the transfer of a phosphate from ATP to creatine when the muscle cell is at rest. It catalyzes the reverse reaction when the muscle is contracting and the cell needs more ATP. creatine + ATP = phosphocreatine + ADP Phosphocreatine serves as an energy phosphate reservoir and is important in heart and muscle (areas with high energy demand). How soon can it be detected? The CK-MB isozyme is first seen at 4 hours after an acute MI and reaches a peak between 12-36 hours. Three isozymes exist: CK-MM, CK-MB, and CK-BB (B=brain, M=muscle). The heart produces a much higher percentage of CK-MB than other tissues. How specific is it for diagnosis? CK-MB is produced mainly by the heart. When the CK-MB is 5% or more of the Total CK, the test is specific for a heart attack. The other isozymes: CK-MM- indicates possible skeletal muscle damage, CK-BB indicates possible brain damage cTN-T (cardiac isozyme of troponin-T): Normal cell functions? Troponin is involved in the regulation of striated and cardiac muscle contraction. Troponin (Tn) is a heterotrimeric protein with three different subunits troponin T (Tn-T), troponin I (Tn-I) and troponin C (Tn-C). The small c in front of TN, i.e., cTN, designates cardiac as compared to skTN for skeletal muscle. How soon can it be detected? It is typically detected in an acute case within 5 hours after the onset of symptoms, is positive in most cases within 8 hours, and has 100% sensitivity at 10-12 hours. This is a little slower compared to myoglobin and CK-MB. How specific is it for diagnosis? cTN-T is unique to cardiac muscle and detection with antibodies is very specific.

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26. Concerning Amy Lloyd: What is an amyloid protein? What are the monomer units of the polymer in amyloidosis/AL? What secondary structure is found in amyloid fibers? What is an M-protein and why is it detected as a sharp peak upon serum protein electrophoresis? Amyloid (amyloid protein) is the extracellular deposition of pathologic insoluble fibrillar proteins, which can cause damage to the area they are deposited in. The term amyloid is a misnomer, as it was originally thought that the fibrils were starch like in nature. Amyloidosis/AL (A = amyloidosis, L = light chain) is where the amyloid is derived from the monomer units: immunoglobulin light chains, principally from the variable region. The secondary structure of the amyloid fibers consists of repeated Beta-sheets (see figure 7.16). The M in M-protein stands for monoclonal. M-protein is produced by an abnormal division of a single type of cell, i.e., monoclonal cell division. In this case, an antibody secreting cell. All of the cells are overproducing a single type of protein. M-proteins produce a sharp narrow peak on electrophoresis because the proteins are all identical so all have the same charge. A normal mixture of serum proteins yields many types of proteins each with their own charge and mobility so there is no peak upon electrophoresis. Extra: The M-protein consists of a single homogenous type of immunoglobulin produced by a single clone of antibodysecreting cells (plasma cells which are from B-Lymphocytes) in the bone marrow. Therefore it is known as "monoclonal", cloned proteins stemming from one cancerous cell.

Top 27. Concerning Di Abietes, What is HbA1c? How is it made and what does it measure? HbA1c is the percent hemoglobin that has been glycosylated. HbA1c is made spontaneously in the blood from hemoglobin and glucose. A molecule of glucose is bound nonenzymatically and irreversibly to the amino terminus of a beta chain of hemoglobin. Since the concentration of hemoglobin and the temperature of the blood are constant, the percent of HbA1c depends only upon the concentration of glucose in the blood. The glucose stays attached to the hemoglobin until the red blood cell is destroyed at about 120 days HbA1c measures the average glucose in the blood for the past 6-8 weeks. The ADA normal range of HbA1c for a normal person is 5.8-7.2%. The ADA upper level for preventing most pathology in a diabetic is 7% Advantages: A patient is not required to fast before the HbA1c test. The patient cannot manipulate the value within a few days of an office visit.

Extra information: The glucose in your blood and intercellular (interstitial)space attach chemically to any proteins present and stay attached permanently. This is thought to be a major cause of the long term pathologies associated with diabetes. Below is an HbA1c to Average Blood Glucose conversion table: HbA1c %- Average Blood Glucose (mg/dl) 4% ---------65 mg/dl, 5% ------- 100 mg/dl, 6% ------- 125 mg/dl, 7% ------- 154 mg/dl, 8% ------- 183 mg/dl, 9% ------- 212 mg/dl, 10% ------ 240 mg/dl, 11% ------ 269 mg/dl, 12% ------ 298 mg/dl, 13% ------ 326 mg/dl, 14% ------ 352 mg/dl,

Top Keywords: ACTIVATION ENERGY BARRIER- The energy the must be put into the substrates of a reaction in order for the reaction to proceed in the forward direction. More in Chapter 8, Fig 8.7. AMYLOID DEPOSITS- extracellular deposits of pathological insoluble fiber proteins. AMYLOIDOSIS/AL- disease characterized by amyloid deposits formed from the varible regions of immunoglobulin Lchains. ANTIPARALLEL B-STRANDS- occurs when the amino terminal to carboxy terminal direction of one strand is in the opposite direction of a second beta-strand to which it is bound. APOPROTEIN is a protein before it is bound to a cofactor (metal ion, coenzyme) necessary for the proteins function. B-SHEETS are formed from beta-strands and account for about 1/3 of all secondary structure. BENCE-JONES PROTEINS are relatively small (MW < 25-50,000 kDa) monoclonal proteins found in blood or urine. BEND nonrepetitive, irregular secondary structure of proteins. CASCADE A series of reactions occurs as a result of a single reaction. Abnormal prion template denatures many normal prions. CHEMICAL MODIFICATION Anything that disrupts the normal conformation of a protein and results in loss of function. CK-MB muscle-brain CK unique to heart, measured to diagnose heart attack. CONFORMATION position of AA side chains in 3D space, determines function of protein.

CONSTANT DOMAINS the domains of h- and L-chains of immunoglobins that do not vary and do not bind to antigen. DOMAIN A stable conformation in a protein that can exist without the rest of the protein. It may be a portion of the tertiary structure or the entire tertiary structure of the protein. DISULFIDE BOND R-S-S-R, critical for holding together & stabilizing tertiary and quaternary structures. FIBRILLAR PROTIENS proteins that appear as small slender fibers or filaments. FOLDS are a description of a domain or tertiary structure of a protein that places an emphasis on how the secondary structure folded into the final conformation. G-GLOBULINS (Gama-globulins, IgG) are one of the five major classes of human immunoglobulins. They contain 2 light chains and 2 heavy chains held in their tertiary structure by hydrogen bonds, salt bonds, hydrophobic interactions, and disulfide bonds. GLYCOSYLATED HEMOGLOBIN- HbA1C is HbA covalently bound to glucose. Its rate of formation is directly proportional to the amount of blood glucose. (6-7% normal range) H CHAINS two heavy chains combine with two light chains to form an immunoglobulin. All H Chains contain constant domains and a variable domain. HbA1C (glycosylated hemoglobin)is HbA covalently bound to glucose. Its rate of formation is directly proportional to the amount of blood glucose. (6-7% normal range) HEMOGLOBIN Hemoglobin ( Hb ) is the oxygen binding protein found in Red Blood Cells (RBCs). It is a tetramer (2 alpha & 2 beta chains ) and each subunit contains an oxygen binding heme. A sigmoidal curve results from cooperativity. HOMOLOGS Two domains or proteins that share the same ancestry are homologous. The have the same, or very similar fold structure, with the same or different function. HEAT SHOCK PROTEINS help proteins fold into their native conformation. Without heat shock proteins, many newly synthesized proteins would fold incorrectly. HEME Heme is the deep red, oxygen-carrying, nonprotein, ferrous (Fe2+ component of hemoglobin. The ferrous ion binds oxygen reversibly in a hydrophobic pocket formed by Hb. HISTIDINE Two histidine R-groups are bound to Fe2+ in hemoglobin. When oxygen binds, it replaces one of the histidine R-groups and pulls the Fe2+ into the plane of the ring. This pulls the second R-group and changes the conformation of the protein. HYDROPHOBIC BINDING POCKET- A hydrophobic pocket of myoglobin & hemoglobin that protects the Fe2+ from oxidation to Fe3+. That is, the oxidation of hemoglobin to methemoglobin. Methemoglobin cannot carry oxygen. Ka is the association constant for a binding site on a protein. :Ka = [Ligand-Protein complex] / {[Ligand] [Protein]} Kd is the dissociation constant for a binding site on a protein. :Kd = 1/Ka L CHAINS two heavy chains combine with two light chains to form an immunoglobulin. All L Chains contain a constant domain and a variable domain. LIGANDS anything that binds to a protein LOOP nonrepetitive, irregular secondary structure M-COMPONENT (M PROTEIN) Electrophoresis of a monoclonal protein yields a single sharp peak containing one homogenous immunoglobulin or portion of an immunoglobulin. Usually produced by a clone of cancer cells in the bone marrow. MOTIF arrangements of secondary structures related to each other (i.e. beta-alpha-loop-beta-alpha-loop). Cannot exist alone and not as large as a domain. MYOGLOBIN- oxygen binding monomer protein, one oxygen binding site, all alpha helices, hyperbolic curve. NONENZYMATIC GLYCOSYLATION- glucose reacts spontaneously, without the help of an enzyme, to form a covalent bond with a protein. (irreversible reaction) pH Changes in pH can change the conformation of a protein and affect their enzyme activity and solubility PARALLEL STRANDS- parallel beta-strands occurs when the amino terminal to carboxy terminal direction of one strand is in the same direction as a second beta-strand to which it is bound.

POSITIVE COOPERATIVITY Occurs when the binding of oxygen, a substrate or ligand at one binding site causes a change in conformation that results in increased binding at a second site. A plot of oxygen binding versus oxygen pressure yields a sigmoidal curve. POST TRANSLATIONAL- Occurs after translation, i.e., after peptide bond formation. PRIONS proteinaceous infectious agents are proteins that catalyze the conversion of PrPc into PrPsc. PrPc- normal conformation of protein prion. PrPsc- disease conformation of prion, enriched beta sheet structurescrapies in sheep (mad cow disease). PROSTHETIC GROUP any ligand necessary for function that binds very tightly and will not dissociate from the protein. The ligand can be a metal ion, a coenzyme, heme, etc.) PRIMARY STRUCTURE linear sequence of AAs in polypeptide chain. Written from the N-terminus to the C-terminus. R (RELAXED) STATE the form of hemoglobin that has a higher affinity to oxygen. QUATERNARY STRUCTURE combination of two or more tertiary structures [(-mers) homo- or hetero- (i.e. homodimer)] SALT BRIDGE (salt bond) Is the ionic attraction between the negative carboxyl on the R-group of aspartate or glutamate and the positive R-group on histidine, arginine, or lysine. Proteins can also form a salt boon using the alpha-carboxyl of the C-terminus and the alpha-amino of the N-terminus. SECONDARY STRUCTURE Is the three dimensional form of local (short) segments of the polypeptide chain into alpha-helices, beta-sheets, and nonrepetitive Secondary Structure (bends, loops, turns) SERUM PROTEIN ELECTROPHORESIS the separation in an electric field of the various proteins in the serum according to their net charge. Proteins with the most negative charge move the fastest toward the anode (+ electrode). Sharp peaks indicate M-protein. SIGMOIDAL S-shaped curve demonstrating cooperation. One example is oxygen saturation graph. SOLVENT A substance in which another substance is dissolved. usually water but sometime lipid or lipid membrane. TEMPERATURE measures the degree of hotness. A measure of the average kinetic energy of the particles in a sample of matter. High temperature (high kinetic energy)can disrupts the bonds holding proteins together and cause denaturation. STRUCTURAL DOMAIN Same as the strict definition of domain: A stable conformation in a protein that can exist without the rest of the protein. It may be a portion of the tertiary structure or the entire tertiary structure of the protein. It has a function, i.e., can perform a physical or chemical task. T (TENSE) STATE the form of hemoglobin that has a lower affinity to oxygen. TERTIARY STRUCTURE folding patterns of 2* structures into 3-D conformations cTN-T is cardiac troponin-T subunit ( c = cardiac, TN = troponin, T is one of three subunits of the troponin molecule). The protein is unique to heart tissue so is specific for death of heart cells. TURN nonrepetitive, irregular secondary structure. VARIABLE DOMAINS- portions of H- and L-chains of immunoglobins that vary to bind to specific antigens. Top gyonuschot@une.edu