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 In this technique, the internal pressure of the cell is increased and suddenly the pressure is withdrawn which causes the rupture of plasma membrane.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)