Saturday, 26 January 2013

Biological oxidation


Many metabolic reactions in the living systems are oxidation-reduction reactions.
Chemically,
Oxidation is defined as addition of oxygen or removal of electrons.
Reduction is defined as removal of Oxygen or gain of electrons Oxidation is always accompanied by reduction of an electron acceptor. These reactions are also known as redox reactions.
In redox reactions free energy change is proportionate to the tendency of reactants to donate or accept electrons. Hence, the change in the free energy can be expressed as redox potential.
Redox reactions can be divided into half reactions. A half reaction consists of an electron donor and its conjugate electron acceptor.
Eg. Fe3+ + Cu+ ______________> Fe2+ + Cu2+ can be divided as

Fe3+ + e- <______________> Fe2+ (reduction)
Cu+ <__________________> Cu2+ + e_ (oxidation)
For electrons to be trasferred both half reactions should occur simultaneously. e- are half reaction's common intermediate.
The two half reactions of a redox reaction, each consisting of a conjugate redox pair, can be physically separated to form an electrochemical cell.
In such a device each half reaction takes place in separate half cell, and e- are passed between half cells as an electric current in the wire connecting their two electrodes. A salt bridge completes the electric circuit by providing a conduit for ions to migrate and there by maintaining neutrality.
The free energy of a redox reaction can be determined by measuring the voltage difference between its two half cells.
The equation to measure redox potential is formulated by Walther Nernst in 1881.
Consider
Aoxn+ + Bred <________________> Ared + Boxn+
in which n electrons per mole of reactants are transferred from reductant ( Bred ) to oxidant ( Aoxn ). The equation for the redox potential can be written as
G= ∆G0' + RT ln ([ Ared ][Boxn+ ] / [ Aoxn+]] [ Bred ])
G= -nF∆E

∆E=∆E0' - RT/nF In([ Ared ][Boxn+ ] / [ Aoxn+]] [ Bred ])

where E= reduction potential
E= Electromotive force
( electron pressure that the electrochemical cell exerts)
The reduction potential when all components are in their standard state is called standard reduction potential.
The redox potential of a redox couple is estimated by measuring the EMF of sample half cell connected to a standard half cell. The sample half cell contains 1molar solution each of the reductant and oxidant. The reference half cell has 1M H+ solution in equilibrium with hydrogen gas at 1atm pressure. The reference half cell has a reduction potential of 0V. But, in biological systems, the redox potential is normally expressed at pH 7.0, at which hydrogen electode is 0.421V
Redox biologically important systems
Negative and positive redox potential:
when a substrate has lower affinity for electrons than hydrogen, it has a negative redox potential.
If the substance has a positive redox potential, it has a higher affinity for electrons than hydrogen.
Thus, NADH, a strong reducing agent has a negative redox otential where as a sterong oxidant like Oxygen has a positive redox potential.

Enzymes involved in oxidation reduction reactions are called oxidoreductases. They can be classified as
      1. Oxidases
      2. Dehydrogenases
  1. Hydroperoxidases
  2. Oxygenases

Oxidases:
Oxidases catalyze the removal of Hydrogen from substrate which is accepted byOxygen. They form mostly water, sometimes Hydrogen peroxide as a reaction product.
                    
AH2  + ½ O------------------>     A  +  H2O   

AH2 + O2  ---------------------> A + H2O2

This group includes Cytochrome oxidase, Tyrosinase, polyphenol oxidase, catachol oxidase and monoamine oxidase etc.
Cytochrome oxidase
It is a haemoprotein having heme as the prosthetic group. It is the terminal component of electron transport chain, and transfers electrons (obtained from the oxidation of substrate molecules by dehydrogenases) to the their final acceptor, Oxygen.
The enzyme is poisoned by Carbon monoxide, cyanide and Hydrogen sulfide. It used to be termed as cytochrome a3 which is now termed as cytochrome aa3. It contains two molecules of heme, each having one Fe atom that oscillates between Fe3+ and Fe2+. It also contains two atoms of Copper.

Some oxidases are flavo proteins. They contain FMN ( Flavin mono nucleotide) or FAD (Flavin adenine dinucleotide) as prosthetic groups. Riboflavin is required for the formation of FMN and FAD in body.
Examples of flavoprotein enzymes are L-aminoacid oxidases, xanthine oxidase and aldehyde dehydrogenase

Dehydrogenases:
these enzymes catalyze the removal of hydrogen from a substrate but oxygen can not act as the Hydrogen acceptor. They catalyze the reversible transfer of Hydrogen from one substrate to another thus bringing abou oxidation and reductions. The often require co enzymes as acceptors of Hydrogen atoms.
A large number of enzymes belong to this group.
NAD+ (Nicotinamide adenine dinucleotide) dependent dehydrogenases:
NAD+ is derived from nicotinic acid, a member of vitamin B-complex. When NAD+ accepts two Hydrogen atoms, one f the hydrogen atom is removed from the substrate as such. The other Hydrogen atom is split into one Hydrogen ion and one electron. The elctron is accepted by NAD+ to neutralize the positive charge on the co enzyme molecule. The remaining Hydrogen ion is released into the surrounding medium.
H2 -----------------> H + H+ + e-
AH2 + NAD+ -----------------> A + NADH +H+

NAD+ linked dehydrogenases are
  • Glyceraldehyde-3- phosphate dehydrogenase
  • Isocitrate dehydrogenase
  • Malate dehydrogenase
  • Glutamate dehydrogenase
  • beta hydroxy acyl co A dehydrogenase
  • Pyruvate dehydrogenase
  • α keto glutarate dehydrogenase

NADP+ (Nicotinamide adenine dinucleotide phosphate) linked dehydrogenases:
they take part in reductive biosynthetic reactions like extra mitochondria pathway of fattyacid synthesis and steroid synthesis. They aso articipate in pentose phosphate pathway.
Eg. HMG coA reductase, enoyl reductase

FAD linked dehydrogenases:
Unlike NADP+ and NAD+ dehydrogenases, FAD accepts both the Hydrogen atoms.
eg. Succinate dehydrogenase, acyl coA dehydrogenase

Cytochromes:
All the cytochromes of electron transport chain except cytochrome aa3 belong to this group.
All cytochromes are heme proteins having iron atom.

Hydroperoxidases:
They use Hydrogen peroxide and any organic peroxide as substrate. Two types of enzymes fall in this category.
  1. Peroxidases
  2. catalases
Hydroperoxidases protect the body against harmful peroxidases. Accumulation of peroxides leads to the generation of free radicals which may cause cancer and atherosclerosis.

Peroxidases: they reduce peroxides using various electron acceptors such as ascorbate, quinones, and cytochrome C. The reaction catalyzed by peroxides is complex but the overall reaction is as follows:
H2O2 + AH2 ------Peroxidase-----> 2H2O
Examples of peroxidases are glutathione peroxidase in RBC which contains Selenium as prosthetic group, leucocyte peroxidase and Horse radish peroxidase.

Catalases: It is a heme protein containing four heme groups. Catalases use H2O2 as both electron donor and acceptor.
2H2O ------Catalase------> 2H2O + O2
Caalase is fund in blood, bone marrow, mucus membrane, kidney and liver. It destructs the H2O2 found by the action of oxidases.
Peroxisomes are subcellular organelles having both oxidase and catalase activity.

Oxygenases:
This group of enzymes catalyze direct direct transfer and incorporation of Oxygen into the substrate molecule.
The catalytic reaction of oxygenase occurs in two steps:
  1. Oxygen is bound to enzyme at active site
  2. the bound oxygen is transferred to substrate
Oxygenases may be subdivided into
a. monooxygenases
b. dioxygenases

a. Monooxygenase:
They are also known as mixed function oxidases. They catalyze the incorporation of one atom of oxygen, while the other atom is reduced to H2O. These enzymes are also called Hydroxylases because OH group is incorporated into the substrate. NADPH usually provides reducing equivalents.

 
Examples of monooxygenases are
  1. phenyl alanine hydroxylase,
  2. hydroxylase,
  3. tryptophan hydroxylase
  4. Nitricoxide synthase
  5. Mitochondrial cytochrome P 450 monooxygenase
    450 denotes that it absorbs light at 450nm, when the heme combines with Carbon Monoxide. It is required for steroid hydroxylation in adrenal cortex, testis and ovary
b. Dioxygenases:
They incorporate both atoms of molecular Oxygen into the substrate.
A + O2 ----------------->AO2
Superoxide dismutase:
Transfer of single electron to O2 generates Superoxide free radical which is potentially damaging by giving rise to free radical chain reaction. The enzyme superoxide dismutase is responsible for the removal of superoxide anion.

The enzyme occurs in all major tissues in the mitochondria and cytosol.

Sunday, 13 January 2013

BIOENERGETICS

BIOENERGETICS

The normal activities of living organisms demand constant input of energy. Even at rest, organisms devote considerable portion of their biochemical apparatus to the acquisition and utilization of energy.
Bioenergetics, also known as biochemical thermodynamics is the study of energy changes accompanying biochemical reactions.
As the biological systems are isothermic in nature, they use chemical energy for the energy requirements of living system.

Basic terms:

Free energy (Δ G): ( Also known as Gibb's free energy)
Free energy is the useful energy in a system.
It is the portion of the total energy change in a system which is available for doing work. It is also known as chemical potential.

Entropy (S):
Entropy is the degree of randomness or disorder of a system. It varies with temperature. 
For example incerease in temperature causes increased disorderness.
Units of S are J.K-1.

Enthalpy:
Enthalpy is the heat content of a system

standard free energy change (ΔG0'):
It is the free energy change under standard conditions.
Standard conditions: pH is 7.0 ans reactant concentration is 1.0 mol/L
Formula for standard free energy change is

ΔG0= −RT ln K ′eq
                                                                                      where R is gas constant
                                                                                                 T is absolute temparature
 
Biological systems obey laws of thermodynamics.

First law of thermodynamics: Energy is conserved
System: The part of the Universe chosen for observation
Surroundings: The rest of the universe
The first law of thermodynamics states that the total energy of a system, including its surroundings, remains constant. It can be neither created nor destroyed. It can be converted from one form to another.
In living systems, chemical energy is converted to heat, radiant or mechanical energy.

ΔE= Q-W
                                                                            where Q is heat absorbed by the system
                                                        W is work done

Second law of thermodynamics:
It states that the total entropy of a system must increase if a process is to occur spontaneously.


Under conditions of constant temperature and pressure, the relationship between the free energy change (ΔG0) of a reacting system and the change in entropy(ΔS) is expressed in following equation, which combines two laws of thermodynamics.
ΔG= ΔH-TΔS
                                                                                 where ΔH = enthalpy
                                                                                                                    T= absolute temperature
In biological systems, ΔH is equal to ΔE
ΔG= ΔE-TΔS
where ΔE= change in internal energy
From the above equation, one can determine the type of reaction .i.e. whether it is exergonic, endergonic or at equilibium.
  • If ΔG is negative, the reaction proceeds spontaneously, and it is exergonic
  • if ΔG is positive, the reaction requires free energy to proceed and it is endergonic
  • If ΔG is zero, the system is at equilibrium and no net change takes place.

In living systems, endergonic processes are coupled with exergonic processes the vital processes like synthetic reactions, muscular contraction, active tansport etc obtain enegy by chemical linkage or coupling to oxidative reactions.
This type of coupling can be represented as

In this, conversion of metabolite A to B occurs with release of energy. This is coupled to another reaction which requires free energy to convert C to D.

In practice, endergonic process must be a component of coupled exergonic-endergonic system.
Usually, the exergonic reactions are catabolic reactions ( breakdown or degradation of molecules) where as endergonic reactions are synthetic or anabolic reactions. Metabolism constitutes anabolic and catabolic reactions in a well coordinated manner.

Mechanism of coupling:.
Mechanism of coupling of exergonic and endergonic reactions may occur in two ways.
  1. By formation of a common obligatory intermediate
  2. Synthesis of high energy compound

1.Formation of a common intermediate:
In this mechanism, a common intermediate is formed, which takes part in both reactions. The intermediate should be structurally related to products and reactants.
A+C ---> I -----> B+D
Examples for this kind of coupling are dehydrogenation reactions, which are coupled to hydrogenations by intermediate carrier.

2. Synthesis of high energy compound:
In this mechanism, a compound of high energy potential is formed during exergonic reaction and it is incorporated into endergonic reaction, thus free energy is transferred from exergonic to endergonic pathways.
As the compound of high potential energy need not to be sructurally related to reactants and products, free energy can be transformed from vide range of exergonic reactions to vide range of endergonic reactions.
The high energy intermediates are usually phosphates and sulfur compounds. These compounds when hydrolyzed release energy in a large quantity. The high energy bond is designated by the symbol ~.

High energy phosphates- ATP:
In living cells, the principal high energy intermediate or carrier is adenosine triphosphate. It is also known as universal currency of energy. ATP is a nucleoside triphosphate containing adenine, ribose and three phosphate groups. It is usually complexed with Mg2+.
The hydrolysis of ATP releases -30.5KJ/mol or -7.3 KCal/mol of energy.
ATP releases energy by donating a phosphate group, and forming ADP. Likewise, ADP accepts a high energy phosphate group to form ATP. (High energy phosphate is designated as
Interconversion of ATP and ADP is catalyzed by the  enzyme Adenylyl kinase.
ATP is continually hydrolyzed and regenerated. An average person at rest consumes and regenerates ATP at a rate of approximately 3 molecules/sec.

Depending on the energy produced by different phosphates they can be classified as
  1. Low energy phosphates They have ΔG0values are smaller than that of ATP.
  2. High energy phosphates The ΔG0value is higher than that of ATP.
standard free energy of some biologically important phosphates



Other high energy compounds are Coenzyme A (eg. Acetyl CoA), acyl carrier protein, aminoacid esters, S-adenosyl methionine, UDP Glucose, PRPP (5' phospho ribosyl-1-pyrophosphate).






Saturday, 5 January 2013

Biochemistry inroduction, Cell fractionation

Biochemistry:

Biochemistry can be defined as the science of the chemical basis of life. It describes the structure, organization and function of cells in molecular terms.

Applications of Biochemistry:
Almost all life sciences require the knowledge of biochemistry, which include Genetics, Physiology, agriculture, medicine, clinical chemistry, nutrition, pathology, immunology, pharmacology, pathology and toxicology.
  1. Genetics: Structure and functions of nucleic acids is the basic of genetics
  2. Physiology: almost all the physiological functions of the body are associated with biochemistry
  3. Immunology: Immunology is a field which requires a number of biochemical techniques
  4. Pharmacology requires a sound knowledge of biochemistry and biochemical processes
  5. Toxicology: Poisons act on biochemical processes
  6. Pathology: Biochemical processes are required for the study of different diseases


Since,Cell is the structural and functional unit of living systems, biochemistry can be described as the science concerned with studying various molecules that occur in living cells and organisms with their chemical reactions.
To study all the chemical processes associated with living cells, it is necessary to isolate biomolecules and larger components and investigate their structure and function. To separate the organelles from the cytosol and from each other Albert Claude, Christian de Duve and George Palade developed methods for cell fractionation.

The steps involved in cell fractionation are:
      1. Homogenization
      2. Separation
      3. Identification

Homogenization:
In the first step of cell fractionation, cells are tissues are disrupted by gentle homogenization. In this procedure, plasma membrane will be ruptured but the other organelles will remain intact.
This can be done
a. by suspending the cells or tissue in isotonic sucrose solution and breaking them by homogenization. The buffer used for preparing isotonoic sucrose solution is TKMg which contains Tris Hcl, MgCl2 and KCl.
b. by subjecting them to high pressure(French press or Nitrogen Bomb). In this technique, the internal pressure of the cell is increased and suddenly the pressure is withdrawn which causes the rupture of plasma membrane.
These steps are carried out at 40C in order to reduce the enzymatic degradation of the cell constituents.

Separation:
Separation of cellular organelles is done by centrifugation of the homogenate.
This can be done by two methods.
  1. Differential centrifugation
  2. Density gradient centrifugation
Differential centrifugation:
in differential centrifugation, subcellular components are separated on the basis of their size. Centrifugation is done at different velocities, by gradually increasing the velocity of centrifugation. Different organelles sediment at different speeds.


These organelles are further purified by density gradient (isopycnic) centrifugation.

Density gradient centrifugation:
In this procedure organelles are separated on the basis of their density. In this procedure, a centrifuge tube is filled with a solution whose density increases from top to bottom. Solution which is used in this procedure can be of sucrose or CsCl2. When a mixture of organelles is placed on top of the density gradient and the tube is centrifuged at high speed, individual organelles sediment until their density matches that in the gradient. Each layer can be collected separately. 


Identification:
Isolated organelles can be identified by the presence of marker enzymes. Marker enzymes are the enzymes which are localized exclusively in the target organelle.

Examples of marker enzymes:

Nuclei
DNA Polymerase, Nicotinamide nucleotide adenyl transferase
Mitochondria
Succinate dehydrogenase, Cytochrome C oxidase
Endoplasmic reticulum
Glucose 6 phosphatase
Lysosome
Acid Phosphatase, Ribonuclease
Peroxisome
Catalase, Urate oxidase
Plasma membrane
5' nucleotidase
Cytosol
Glucose 6 phosphate dehydrogenase, lactate dehydrogenase, 6- phosphofructo kinase
Golgi complex
Galactosyl transferase


Cell:
The smallest organisms consists of single cells where as larger organisms which are multicellular contain different types of cells. Although, they differ in their function, they share some fundamental properties which can be studied at the biochemical level.

Major Features of a Typical Animal Cell

1.Extracellular matrix:
The surfaces of animal cells are covered with a flexible and sticky layer of carbohydrates, proteins, and lipids. This complex coating is cell-specific, serves in complex cell – cell recognition and communication,creates cell adhesion, and provides a protective outer layer.

2.Cell membrane (plasma membrane):
It is Roughly 50:50 lipid:protein as a 5-nm-thick continuous sheet of lipid bilayer in which a variety of proteins are embedded. The plasma membrane is a selectively permeable outer boundary of the cell, containing specific systems—pumps, channels,transporters—for the exchange of nutrients and other materials with the environment. Important enzymes are also located here.

3.Nucleus :
The nucleus is separated from the cytosol by a double membrane, the nuclear envelope (continuous with endoplamic reticulum).The membrane is punctuated by a large number of nuclear pores, which are composed of proteins that permit diffusion of small molecules and limited diffusion of larger molecules. Very large molecules also diffuse across if they possess the correct ‘identifying signal’.
DNA is present within the nucleus which possesses the information required for the synthesis of almost all the
proteins in the cell (except proteins produced in mitochondria). The DNA is complexed with basic proteins(histones) to form chromatin fibers, the material from which chromosomes are made. 
Nucleolus which is visible in the nuclei of most cells, especially those actively synthesising protein, consists
of a mass of incomplete ribosome particles and DNA molecules that code for ribosomal RNA: this is the site of synthesis of the ribosomal subunits.
The nucleus is the repository of genetic information encoded in DNA and organized into chromosomes. During mitosis, the chromosomes are replicated and transmitted to the daughter cells. The genetic information of DNA is transcribed into RNA in the nucleus and passes into the cytosol where it is translated into protein by ribosomes.

4.Mitochondria:
Mitochondria are organelles surrounded by two membranes that differ markedly in their protein and lipid composition. The inner membrane and its interior volume, the matrix, contain many important enzymes of energy metabolism. Mitochondria are about the size of bacteria approximately of 1 micro meter. Cells contain hundreds of mitochondria, which collectively occupy about one-fifth of the cell volume.
Mitochondria are the power plants of eukaryotic cells where carbohydrates, fats, and amino acids are oxidized to CO2 and H2O. The energy released is trapped as high-energy phosphate bonds in ATP.

5 Golgi apparatus:
A system of flattened membrane-bounded vesicles often stacked into a complex. It has cis and trans aces. Cis- face of golgi faces towards the centre of the cell. Numerous small vesicles are found peripheral to the Golgi and contain secretory material packaged by the Golgi.
Golgi apparatus is Involved in the packaging and processing of macromolecules( eg. proteins) for secretion and for delivery to other cellular compartments.

6.Endoplasmic reticulum:
These are flattened sacs, tubes, and sheets of internal membrane extending throughout cytoplasm of the cell and enclosing a large interconnecting series of volumes called cisternae. The ER membrane is continuous with the outer membrane of the nuclear envelope. Portions of the sheet like areas of the ER are studded with ribosomes giving rise to rough ER. 
It has four main functions:
i.  Synthesis of those proteins that are destined for incorporation into cellular membranes or for export from the cell. Transport of those proteins that are destined for cell membranes or for release from the cell is achieved through vesicles that pinch off from the endoplasmic reticulum and fuse with membranes of the Golgi.
ii. Synthesis of phospholipids and steroids.
iii. Hydroxylation (addition of an –OH group) of compounds that are toxic or waste products, which renders them more water soluble, hence they are more rapidly excreted. These are known as detoxification reactions.
iv. Storage of Ca2+ ions at a concentration 10 000 times greater than in the cytosol (i.e. similar to that in the extracellular fl uid, about 10−3 mol/L). It is the release of some of these ions that acts as a signalling process in the cell. For example, stimulation of contraction of muscle by a nerve depends upon Ca2+ ion release from the reticulum into the cytosol of the muscle cell.

7.Lysosomes:
Lysosomes are vesicles of 0.2–0.5 micro meter in diameter, bounded by a single membrane.They are formed by budding from the Golgi apparatus.The pH within this organelle is very low (about 5.0) and the catalytic
activities of the enzymes, within it, are highest at this pH. They contain hydrolytic enzymes such as proteases and nucleases.
The enzymes degrade a number of compounds:
• Proteins taken up from outside the cell or those damaged within the cell.
• Particles, including bacteria, taken up from the environment.
• Damaged or senescent organelles (e.g. mitochondria).


8.Peroxisomes:
Like lysosomes, peroxisomes are 0.2–0.5 micro meters single-membrane–bounded vesicles. contain a variety of oxidative enzymes that use molecular oxygen and generate peroxides. They are formed by budding from the smooth ER.
Peroxisomes act to oxidize certain nutrients, such as amino acids. In doing so, they form potentially toxic hydrogen peroxide, H2O2, and then decompose it to water and O2 by way of the peroxide-cleaving enzyme, catalase.

9. Ribosomes:
 Unlike the organelles described above, ribosomes have no membrane but are aggregates of ribonucleic acid (RNA) and protein. Each ribosome consists of two subunits: a large and a smaller one.
Most of the protein synthesis in a cell takes place within or upon the ribosomes. They bring together messenger RNA (mRNA) and the components required for protein synthesis.
 
10.Cytoskeleton:
The cytoskeleton is composed of a network of protein filaments: actin filaments(or microfilaments), 7 nm in diameter; intermediate filaments, 8–10 nm; and microtubules, 25 nm. These filaments interact in establishing the structure and functions of the cytoskeleton. This interacting network of protein filaments gives structure and organization to the cytoplasm.

 

Biochemical techniques used for the study of various biochemical process are:
I. Methods for Separating and Purifying Biomolecules:
1. Salt fractionation (eg, precipitation of proteins with ammonium sulfate)
2. Chromatography: Paper; ion exchange; affinity; thin-layer;gas-liquid; high-pressure liquid; gel filtration
3. Electrophoresis: Paper; high-voltage; agarose; cellulose acetate; starch gel; polyacrylamide gel; SDS- polyacrylamide gel
4. Ultracentrifugation
II. Methods for Determining Biomolecular Structures
1.Elemental analysis
2.UV, visible, infrared, and NMR spectroscopy
3.Use of acid or alkaline hydrolysis to degrade the biomolecule
4.under study into its basic constituents
5.Use of a battery of enzymes of known specificity to degrade the biomolecule under study (eg,proteases, nucleases,glycosidases)
6.Mass spectrometry
7.Specific sequencing methods (eg, for proteins and nucleicacids)
8.X-ray crystallography
III. Preparations for Studying Biochemical Processes
1. Whole animal (includes transgenic animals and animals with gene knockouts)
2. Isolated perfused organ
3.Tissue slice
4. Whole cells
5. Homogenate
6. Isolated cell organelles
7. Subfractionation of organelles
8. Purified metabolites and enzymes
IV.Isolated genes (including polymerase chain reaction and site-directed mutagenesis)