Team 3 Running TBL.docx (hucm-2015-tbl5@googlegroups.com)

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This is a running list of the TBLs for Team 3. :)

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Team 3

TBL1

3. A fat, or a triacylglycerol is an ester of glycerol, where all three hydroxyl groups of glycerol are esterified with fatty acids.  Fatty acids consist of an alkyl chain with a terminal carboxyl group.  Nearly all in humans have an even number of carbon atoms.

The long hydrocarbon chains of the fatty acids allow them to store energy more efficiently, because they do not associate with water in the process.  Triacylglycerols are insoluble in water and do not form hydrogen bonds. A 6-carbon-glucose molecule creates 36-38ATP, on average, providing a ratio of 6ATP/carbon; whereas, an 18 carbon fatty acid produces 147 ATP (8.2ATP/carbon).  

An example of a disulfide bond could be found in the tertiary structure in proteins. Insulin is a common protein capable of disulfide bonding.  Cysteine is the only amino acid able to form disulfide bonds.   Oxidation of thiol group on sidechain.  Cross links the proteins, making the protein rigid, keeping the protein from denaturing.

Thioester:  Acetyl CoA: may be oxidized for energy productions (makes fats or burns them).  Functions to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production.  Also important in biogenic synthesis of the neurotransmitter acetylcholoine.

Thioether:  Biotin: plays a role in gluconeogenesis and is a coenzyme involved in the metabolism of fatty acids and leucine.

TBL 2

3.  (a) Using Henderson Hasselbalch: pH=pKa+log(conjugate base/conjugate acid)

7.4=pKa+log(3.5/1)

pKa=6.856

(b) 3.5/4.5=77.78

(c) Using Henderson Hasselbalch: pH=pKa+log(conjugate base/conjugate acid)

        Stomach: 1.5=3.5 +  log(CB/CA)                

                Ratio of CA/CB is 10^-2=0.01

        Small Intestine: 6=3.5 + log(CB/CA)

                Ratio of CB/CA is 10^2.5=316.23

TBL3 Bioenergetics

Why are some compounds classified as “high energy” compounds? 

“High energy compounds” are compounds that have a high capacity to do work; thereby, they have a large negative ΔG0 value (between -5 to -15 kcal/mole).  The products of hydrolysis are more stable than reactants for high energy compounds.  Generally, molecules with shorter,  multiple (i.e. double and triple bonds), and or phsophoanhydride bonds are considered high energy.  An example of a high energy compounds is ATP.  Some others are 1,3 Bisphosphoglycerate, phosphoenolpyruvate, and creatine phosphate.

Why are products of hydrolysis of high energy compounds more stable than reactants?

Products of hydrolysis of high energy compounds are more stable than the reactants because they are at a lower energy state.  These products have a greater resonance stability; therefore there is less bond strain, due to electrostatic repulsion of oxygen atoms.  During hydrolysis bonds are broken between oxygen and hydrogen, releasing energy and water.  

TBL4

QUESTION: List and describe two examples in which proteomics has been used for clinical applications.  

ANSWER        

Proteomics is the systematic evaluation of changes in the protein constituency of a cell. It is a large scale study of proteins, focusing structure and function. It generates a list of proteins; the ultimate goal is to characterize the information flow through protein pathways.  Proteomics may be used as a biomarker and therapeutic target.  Effective biomarkers must be measurable in accessible body fluid such as serum, urine, or saliva.

Mass spectrometry is used in proteomics; the mass to charge [m/z] ratio versus signal intensity is measured.   Recently, there have been developments for serum-based proteomic pattern diagnostics, which is a new method of diagnosis and disease identification for ovarian cancer detection.  The new concept is that the diagnostic endpoint for ovarian cancer detection is not a single analyte but a proteomic pattern that is composed of many individual proteins, each of which independently cannot differentiate diseased from healthy individuals.

     Additional Info: “Surface-enhanced laser desorption and ionization (SELDI) technology. This type of proteomic analytical tool is a class of mass spectroscopy instrument that is useful in high-throughput proteomic fingerprinting of serum. Using a robotic sample dispenser, 1 µL of serum is applied to the surface of a protein-binding chip. A subset of the proteins in the sample bind to the surface of the chip. The bound proteins are treated with a matrix-assisted laser desorption ionization matrix and are washed and dried. The chip, which contains multiple patient samples, is inserted into a vacuum chamber where it is irradiated with a laser. The laser desorbs the adherent proteins and causes them to be launched as ions. The time of flight (TOF) of the ion before detection by an electrode is a measure of the mass-to-charge (m/z) value of the ion. The ion spectra can be analyzed by computer-assisted tools that classify a subset of the spectra by characteristic patterns of relative intensity.”

The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.

TBL5

QUESTION: . A polypeptide is subjected to the following degradative techniques, resulting in the indicated fragments. What is the sequence of the entire polypeptide?

I.        Cyanogen bromide treatment yields

Fragment 1: Asp-Ile-Lys-Gln-Met

Fragment 2: Lys

Fragment 3: Lys-Phe-Ala-Met

Fragment 4: Tyr-Arg-Gly-Met

II.        Trypsin hydrolysis yields

Fragment 1: Gln-Met-Lys

Fragment 2: Gly-Met-Asp-Ile-Lys

Fragment 3: Phe-Ala-Met-Lys

Fragment 4: Tyr-Arg

        

Prion Question: How does the infectious form of PrP “propagate” itself in its host? (Devlin can help with this, as can your lecture notes).

ANSWER

Cyanogen bromide treatment cleaves methionine on the C-terminus.  Trypsin hydrolysis cleaves Arginine and Lysine on the C-terminus.  

1. Look at number of amino acids in each
2. Look for repetition
3. Look for abnormalities in fragments (i.e. no Met in a fragment for cyanogen bromide treatment)
4. Compare two methods to match amino acids

I.        Cyanogen bromide treatment yields

Fragment 1: Asp-Ile-Lys-Gln-Met

Fragment 2: Lys (end)

Fragment 3: Lys-Phe-Ala-Met

Fragment 4: Tyr-Arg-Gly-Met

II.        Trypsin hydrolysis yields

Fragment 1: Gln-Met-Lys (end?)

Fragment 2: Gly-Met-Asp-Ile-Lys

Fragment 3: Phe-Ala-Met-Lys (end?)

Fragment 4: Tyr-Arg

        

Tyr-Arg-Gly-Met-Asp-Ile-Lys-Gln-Met-Lys-Phe-Ala-Met-Lys

Prions, or protein infectious agents occur in the body in the normal state (α-helix).  However, once the prion transforms to the β-sheet conformation, it becomes infectious.  PrP propagates itself by seeking out proteins similar to itself and changing their structural conformation, causing an accumulation of infectious prions and leading to fatal neurodegenerative diseases.

TBL6

QUESTION

Describe the changes in globin chain production during development. Why would a newborn with sickle cell disease show few signs of the disease (Hint: Think about developmental heterogeneity of hemoglobins)?  Why would a hereditary persistence of HbF ameliorate the severity of sickle cell disease?

ANSWER

 

With the exception of the very first weeks  of embryogenesis, one of the globin chains  is always alpha. A number of variables  influence the nature of the non-alpha  chain in the hemoglobin molecule. The  fetus has a distinct non-alpha chain called  gamma. After birth, a different non-alpha  globin chain, called beta, pairs with the  alpha chain. The combination of two alpha  chains and two non-alpha chains produces  a complete hemoglobin molecule (a total  of four chains per molecule). During development, the globin chain changes from δ (embryonic), to γ (fetal), to β and δ (adult) for the beta type.  The Alpha types change from ζ (embryonic) to α (fetal/adult).  

A newborn with sickle cell disease would show few signs of the disease because they do not have the correct form of hemoglobin to display the disease.  Fetal hemoglobin is 70-90% HbF.  HbF is asymptomatic for β-chain defect; therefore, β-thalassmia and sickle cell disease are not evident, since sickle cell is a β subunit mutation..

Sickle-cell anemia arises from a mutation in the gene for the β chain of human hemoglobin. A hereditary persistence of HbF would ameliorate the severity of sickle cell disease because HbF has a different amino acid sequence, providing a distinctive chain, the gama chain instead of the beta chain, during most of the fetal life. molecules do not participate in the polymerization that occurs between molecules of deoxyHbS (Goldberg, et al., 1978). The gamma-chain lacks the valine at the sixth residue to interact hydrophobically with HbS molecules. HbF has other sequence differences from HbS that impede polymerization of deoxyHbS. Second, higher concentrations of HbF in a cell infer lower concentrations of HbS (Bailey et al, 1991). Polymer formation depends exponentially on the concentration of deoxyHbS (Eaton and Hofrichter, 1995). Each of these effects reduces the number of irreversibly sickle cells (ISC).

TBL7

QUESTION

Draw the structure of the heme prosthetic group. Explain what is the difference between the following terms: porphyrin, heme and hemoglobin? What groups on Hb are responsible for binding iron, oxygen, carbon monoxide, heme, protons, carbon dioxide, 2,3-BPG? What are the major sites of Hb synthesis and degradation in the body? Briefly outline the processes of hemoglobin synthesis and degradation.

ANSWER

Heme Prosthetic Group

 

1. A porphyrin is a heterocyclic molecule composed of four pyrrole (a 5 membered ring with the formula C4H4NH) subunits interconnected at their alpha carbons via methine (=CH) bonds.

 

1. Heme is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. It is aromatic and and is responsible for the red pigment in hemoglobin.

 

1. Hemoglobin is a hetrotetramer (2α2β, normal adult Hb) with a heme in each subunit.

1. Groups on Hb responsible for binding:

1. Iron: Four bonds to nitrogen (from the pyrrole rings), one bond to histidine (F-8), (and the 6th bond is to Oxygen)
2. Oxygen: Binds between heme and distal histidine(Binds to Fe++ at the 6th site and H-bond to distal His (E-7) ).
3. CO: replaces Oxygen and Binds to Fe++ at the 6th site
4. Heme: tightly bounded in a hydrophobic binding pocket(Bind to the F-8 histidine residue). (May also be bonded to a ligand like oxygen or carbon monoxide)
5. 2,3 Biphosphoglyceric acid (2,3 BPG) : binds (ionically) to the lysine and histidine residues within the hemoglobin subunits (binds to a pocket formed by the two beta-globin chains in the center of the tetramer). This binding causes a lower affinity to oxygen, transitioning the hemoglobin from R (oxygenated) to T (deoxygenated) state.
6. CO2: Binds an amino group at an allosteric site.
7. Protons: bind to the iron (Fe2+) increasing the oxidation state to (Fe3+). This type of oxidation can lead to the formation of methemoglobin, which has a higher affinity for oxygen. This increased affinity can eventually lead to tissue hypoxia. However, changes in iron oxidation state do occur normally when the iron is already within the globin molecules. The iron is oxidized so that it will lie in plane within the globin molecules when attached to molecular oxygen (O2).

Briefly outline the processes of hemoglobin synthesis and degradation:

1. Hb synthesis: found in bone marrows cells . First, aminolevulinic acid (ALA) is synthesized using two building blocks: glycine and succinyl CoA. The dehydration of two molecules of ALA forms porphobilinogen. Then, the condensation of four molecules of porphobilinogen results in the formation of uroporphyrinogen. Lastly, a series of decarboxylations and oxidations converts it to heme.

1. Hb degradation: The heme/hb degradation occurs in the Spleen and is phagocytosed to form bilirubin and amino acids. Since bilirubin is only slightly soluble, it is transported to the liver by albumin. In the liver, two molecules of glucuronic acid are added to bilirubin to increase its solubility, which results in bilirubin diglucuronide. It is then excreted into the bile. Bilirubin diglucuronide is hydrolyzed and reduced by bacteria to form stercobilin; which gives feces its brown color.

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