Physical properties of amino acids

• Amino acids are colorless, crystalline substance.
• Most amino acids are tasteless but some are sweet. (E.g. Glycine, Alanine) and some are bitter (Eg. Arginine)
• Amino acids have high melting point (200-300)^oC due to ionic property.
• Solubility:
  • Solubility of amino acids depends upon polarity, iso-electric point, nature of solvent (pH) and temperature.
  • Amino acids are soluble in water and ethanol (i.e. polar solvent) and insoluble in non-polar solvent like benzene, ether etc.
  • Amino acids are insoluble at iso-electric point.
  • Solubility depends upon pH of solvent and temperature.
  • Eg. Tyrosine is soluble in hot water.

• Amphoteric property:
  • Amino acids can act as acid and base due to their dipolar i.e. zwitter ion nature.

• Titration curve of amino acids:
  • Most of the amino acids are monoamino-monocarboxylic acid.
  • When they are in fully protonated form they can be titrated twice.
  • Titration curve is the graph made between pH of amino acids and volume of acid or base added. It is always sigmoidal.
  • pKa= tendency of an acid (-COOH) to loose proton.
  • This tendency decreases 10 folds as pKa value increase by 1 unit.
  • At pKa, there is equimolar concentration of +ve ion and zwitter ion.

Important information from titration curve:

• pKa, pKb and pI value can be calculated.
• All the monoamino-monocarboxylic aminoacids (diproteic) have nearly about pKa and pkb values.
• Buffering zone (pKa+/-1) or (pKb+/-1).
• Glycine: pKa=2.3, pKb=9.6
• Alanine: pKa= 2.26, pKb=9.4
• Aspartic acid: pKa+2.09, pKb=9.82, pKR=3.86
• Histidine: pKa= 1.82, pKb=8.95, pKR=10.53

• Note:
  • The α-carbonyl group of mono-amino-mono carboxylic amino acids is stronger acid than carboxyl group of the aliphatic acid.
  • It is because of amino group and its +ve charge which increase tendency of carboxyl group to dissociate the -ve charge.
  • Similarly, the α amino group of diproteic amino acid is stronger base than amino group of comparable aliphatic amines. It is because of presence of carboxyl group.

• Absorption spectrum:
  • Amino acids absorbs the light at 280nm so the concentration of amino acids can be measured.

• Isomerism in amino acids:
  • Except glycine, all amino acids exists in super impossible mirror image i.e. D and L form.
  • This configuration is given by Emil Fischer.
  • This configuration indicate absolute concentration but not indicate the optical activity of amino acids.
  • – CHO group can be converted into -COOH group so they are compared.
  • -OH group compared with -NH₂ group.
  • -R- group compared with -CH₂OH group.

Chemical properties of amino acids:

• Why chemical properties of amino acid is importance?
• Chemical reactions of amino acids are important:
  • For identification and analysis of amino acids in protein.
  • For identification of amino acid sequences in protein.
  • For identification of specific amino acid residue of native protein that are required for biological functioning e.g. haemoglobin (Histidine has role in it).
  • For chemical modification of amino acids residue in protein molecules to produce change in biological activity.
  • For chemical synthesis of the polypeptides (for medical purpose).

Types of chemical reactions given by amino acids:

• Reaction due to -COOH group.
• Reaction due to -NH₂ group
• Reaction due to both
• Reaction due to R-group

1. Chemical reactions of amino acids due to -COOH group:

2. Chemical reactions of amino acids due to both -COOH and -NH2 group:

3. Chemical reaction of amino acids due to -NH2 group

4. Chemical reaction of amino acids due to functional group:

Functions of amino acids:

• Precursor for synthesis of proteins and polypeptides.
• Used for glucogenic and ketogenic degradation.
• For synthesis of porphyrin.
• For synthesis of melanin: Melanin is complex polymeric structure made up of tyrosine and also may contain tryptophan.
• For synthesis of creatine and creatine phosphate.
• For synthesis of plant hormones (auxin).
• For synthesis of neurotransmitter.
• For synthesis of animal hormones (thyroid- thyroxine)
• For synthesis of lignin, tannin, papaverine (alkaloids).
• For synthesis of vitamins (Ascorbic acid), niacin.
• Synthesis of antibacterial agents (penicillin G).

Properties of amino acids: physical and chemical

The post Properties of amino acids: physical and chemical appeared first on Online Biology Notes.

Classification of lipid on the basis of composition:

I. Simple lipids:

They are further divided into neutral fat and oil and wax.

i. Fat and oil:

• Fat and oil are triglycerides, a triacylglycerol (TAG) in which 3 fatty acids are linked with one glycerol molecule by ester bond.
• TAG containing same type of fatty acids is called simple TAG, whereas those containing different types of fatty acids are called mixed TAG.
• Most naturally occurring fat and oil are mixed type.
• Fatty acid commonly occurring are C₁₆, C₁₈, type i.e. palmitic acid, steric acid and oleic acid.
• Triglyceride occupy 98% of diet lipid. They are the storage form of energy and usually form fat depot.Fat contains mainly saturated fatty acids therefore they are liquid at room temperature.
• Fat and oil are non-polar and hydrophobic in nature because the -OH group of glycerol and -COOH group of fatty acids are involved in ester bond formation and is not available for H-bonding.
• Functions:
  • Fat and oil provide energy in cell. Oxidation of one gram of fat release 9.3kcal energy.
  • They are important source of energy when carbohydrates are unavailable in cell.
  • They have insulating effect during cold.
  • They are stored in seed as fat depot and help during germination of seed.
  • In sperm whale, presence of triglyceride gives buoyancy (upthrust).
  • They are storage form of energy mainly formed in- subcutaneous layer.

ii. Wax:

• R₁-OH (monohydroxy alcohol) + HO-OCR₂ (fatty acids)à R₁-O-C=OR₂ (wax) + H₂O
• CH₃-(CH₂)₂₈-CH₂OH (myricyl alcohol) + CH₃-(CH₂₎₁₄-COOH (palmitic acid) à CH₃-(CH₂)₂₈-CH₂-O-C=O-(CH₂)₁₄-CH₃ (myricyl palmitate) (Bee’s wax).
• Waxes are the ester of fatty acid with high molecular weight monohydroxy alcohol.
• Different types of alcohols and FA are found in different naturally occurring waxes.
• Examples: bee’s wax, myricyl alcohol, palmitic acid, carnauba’s wax, tetracosanol, tetra triacosanol.
• Waxes are more solid and hydrophobic than fat and oil due to large hydrocarbon portion.
• Waxes are secreted by sebaceous gland, preen gland (below feather of bird), bees, sheep’s wool, sperm of whale etc.
• Functions:
  • They act as source of energy in some marine moss. E.g. planktons
  • Due to their water repelling property and smooth textures.
  • They are used for preparation of cosmetic products and boot polish.

II. Complex lipids:

• They are further divided into:
  • Phospholipids
  • Glycolipids

1. Phospholipid:

• They are further divided into two types:
  • Glycerophospholipid
  • Spingophospholipid

i. Glycerophospholipid:

• Glycerophospholipid consists of glycerol in which two fatty acids are linked with -OH group glycerol by ester bond and third -OH group of glycerol is linked with phosphate group which in turn linked with head group substituent.
• Head group substituents are different in different glycerophospholipid.

ii. Spingophospholipids: (Spingomylein):

• Spingophospholipid contain an amino alcohol called spingosine instead of glycerol.
• In spingophospholipid, one fatty acid is linked with -NH₂ group of spingosine by peptide bond and -OH group of spingosine linked with PO₃^—with in turn linked with head group substituents.
• Spingophospholipid (ceramide):
• Head group are different in spingolipid.
• If the head group in choline then it is called spingomylein.

Function of Phospholipids:

• Phospholipids are structural components of cell membrane. They make lipid bilayer of cell membrane.
• Lecithin helps in transport and metabolism of other lipids in animal.
• Lecithin deficiency causes deposition of large amount of fat in liver causing fatty liver.
• Phosphatidyl inositol-4,5-bisphosphate help in regulation of cell structure and metabolism.
• Platelets aggregating factor help in aggregation of platelets and release of serotonin from platelets.
• Helps in enzyme catalysis, ETS cycle.
• Phosphatidyl ethanol amine (cephalin) play important role in cell division, cell fusion etc.
• Phosphatidyl serine is a flippase enzyme.

2. Glycolipids:

• They are of two types:
  • Glyceroglycolipids
  • Spingoglycolipids

i. Glyceroglycolipids:

• In glyceroglycolipid, two fatty acids are linked with glycerol by ester bond and their OH group of glycerol is linked with carbohydrate head group.

ii. Spingoglycolipids:

• It consists of spingosine instead of glycerol in which -NH₂ group is linked with fatty acids by peptide bond and -OH group is linked with carbohydrate head group.
• Head group is different in different spingoglycolipid.

Functions of glycolipids:

• They are structural component of cell membrane.
• O, A, B antigen on RBC surface determine blood group.
• Help in signal transduction.
• Role in growth and tissue differentiation as well as carcinogenic.
• Gangliosides are present in brain (6%).

Sulpholipids:

• They are sulfate ester of glycolipid.
• Found in chloroplast, chromophore of bacteria.

Aminolipids:

• Mostly found in bacteria in outer and inner membrane, it is also called as mololipid. E.g. lipid containing serine.
• -(CH₂)₆-CH₂-CHOH-CH₂-C=O-NH-CHCH₂OH-COOH
• Found in Serectia.
• Lipid containing glycine: iso-3-hydroxyheptadecanoic acid linked to glycine.
• Proteolipid: contain protein attached to lipid.

III. Derived lipids:

• They are the hydrolyzed product of simple and composed lipids with various type of other compounds such as alcohol, ketone, vitamin D, sex-hormone steroid, terpenes, carotenoids.

Steroid:

• Sterane ring (Cyclopentanoperhydrophenanthrene).
• Sterane is parent compound.
• All steroids are derivatives of sterane. They are more hydrophobic than other lipids.
• E.g. cholesterol, sex hormone, Vit.D.

The post Classification of lipid appeared first on Online Biology Notes.

• Oxidation of fatty acid (FA) releases large amount of energy.
• Oxidation of fatty acid occurs in mitochondria cell lacking mitochondria (e.g. RBC) and brain cell (due to blood brain barrier) cannot oxidize FA to release energy.
• Most of the fatty acids are oxidized by beta-oxidation.

Steps in beta-oxidation of fatty acids:

1. Activation of Fatty acid
2. Transport of fatty acyl coA into mitochondria
3. Beta- oxidation

Step I: Activation of fatty acid

• Fatty acid is converted to fatty acyl CoA by thiokinase or fattyacyl CoA synthetase.
• This reaction occurs in cytoplasm and is ATP and Mg²⁺.

Step II: Transport of acetyl coA into mitochondria

• The inner mitochondrial membrane doesn’t permit fatty acids to pass through it.
• The activated FA enter mitochondria through carnitine shutter.
• This occurs in 4 steps:
• Fatty acyl coA is transferred to carnitine to form fatty acyl carnitine. This is catalyzed by enzyme carnitine acyl transferase I (CAT-I) present on outer surface of mitochondria.
• Then acyl carnitine enters into matrix through carnitine shutter.
• Fatty acyl carnitine is converted into fatty acyl CoA by an enzyme carnitine acyl transferase II (CAT-II) formed in inner mitochondrial membrane.
• The carnitine is released and return to cytosol for re-use.

Step III: Beta-oxidation

• For example; Beta-oxidation of saturated FA (palmitic acid):
• Beta-oxidation occurs in cycle and each cycle consists of sequence of four enzymatic reaction:
  • An oxidation step that produce FADH₂
  • A hydration step
  • A second oxidation step that produce NADH+ H⁺
  • A thiolytic cleavage that release a molecule of acetyl coA.
  • The fourth step of beta-oxidation repeated for (n/2-1) times where n=no. of carbon atoms.
• For e.g. Palmitic acid consists of (16/2-1) = 7 beta-oxidation cycle.
• Each beta-oxidation cycle produces 1 FADH₂, 1 NADHA + H⁺ and 1 acetyl CoA.
• Total (for palmitoyl CoA):
  • FADH₂ -7
  • NADH₂ – 7
  • Acetyl CoA – 8
• Total of 7 FADH₂, 7NADH+ H⁺ and 8 acetyl CoA are generated from production of palmitic acid.
• Acetyl CoA enter into TCA and each acetyl CoA release 12 ATP.
• Similarly, FADH₂ and NADH+H⁺ enter ETS cycle to generate 2ATP and 3ATP respectively.
• Therefore,
  • 8 acetyl CoA = 8X12=96
  • 7 NADH₂= 7X2= 14
  • 7 NADH₂ + H⁺ = 7X3 = 21
  • Total———————181 ATP
• Net ATP gain is 129 (131-2).
• This is because 2ATP are utilized during conversion of fatty acid (palmitic acid) into fatty acyl CoA (palmitoyl CoA).

Example: Beta oxidation of Unsaturated fatty acid (Oleic acid) and Polyunsaturated fatty acid (Linoleic acid)

• monounsaturated fatty acid consists of a double bond. Beta oxidation of monounsaturated fatty acid involves most of the reactions same as found in beta oxidation of saturated fatty acid. However, an extra enzyme enoyl coA isomerase is needed.
• similarly, due to presence of two or more double bonds, beta oxidation of poly unsaturated fatty acid requires two additional enzymes- enoyl coA isomerase and 2, 4- dienoyl coA reductase.

Beta Oxidation of fatty acid: steps and examples

The post Beta Oxidation of fatty acid: steps and examples appeared first on Online Biology Notes.

• The process of breaking apart of the cells in order to obtain the desired product which may be intracellular or periplasmic is called cell disruption.
• There are various methods for cell disruption but before employing any method, following factors should be considered before selecting any method.
  • Size of cell sample
  • Toughness of the cell is whether it is bacteria, fungi, plant cell or animal cells.
  • Efficiency of disruption method
  • Stability of the product to the method employed.
  • Easy extraction and purification
  • Whether the cell is biohazardous or not
  • Cost and time
  • Expertise or training required or not.

Methods of cell disruption

Physical methods of cell disruption:

• Mechanical method:
  • Bead will
  • Homogenizer
  • Microfluidizer
  • Sonicator
  • French press/ X-mesh
• Non-mechanical method:
  • Decompression
  • Osmotic shock
  • Thermolysis
  • Freeze thaw
  • Dessication
  • Cell bomb

These Mechanical methods are used to break tough cells.

i. Bead mill:

• The bead will consist of tubular vessel made up of metal or glass within which small beads are kept.
• When vessel rotates, the beads also starts rotating but away from axis.
• The shear force and impact of beads causes disruption of cells.
• Advantages:
  • It is very useful for small sized materials and it doesn’t release harmful aerosols.
  • Bead mill can be carried out in both batch and continuous fashion.
  • Commonly used for disruption of yeast cells and for grinding animal tissues.
• Disadvantages:
  • It produces large amount of heat so thermolabile materials cannot be disrupted.

ii. French press:

• French press consists of a cylinder with orifice at bottom.
• The piston is pressed with hydraulic pressure so that cell suspension exists through small orifice and strike on impact place.
• The impact causes cell disruption.
• It is commonly used for small scale recovery of intracellular materials from bacterial and plant cell.

iii. Sonicator:

• About 50Khz frequency is applied on cell suspension which causes the formation of tiny bubbles within liquid.
• It is very fast method.
• It breaks cells in 30-60 second and yeast cell (2-3mins).
• Disadvantages:
  • Heat generation
  • Noise pollution
  • Expensive process
  • Generate free radicals that might interfere desired product.

iv. Homogenizer:

• Different types of tissue homogenizer are used:
• Pressurized homogenizer
• High pressure homogenizer e.g. APV

v. Ultra sonicator

• APV is high pressure homogenizer used in large scale.
• The pressure is applied through valve pressure, when pressure inside the vessel reaches the atm pressure, cell bursts in this process, no heat is generated because inert gases are used in the vessels.
• This is a gentle technique used for animal cells disruption but not for plant cells and fungi.

vi. Microfluidizer:

• In this method pressure is applied that causes production of small sized particles.
• This method is applied to lab scale.
• Pressure and shear force cause disruption of cell.
• Disadvantages:
  • Expensive method
  • Cannot used for large scale production

II. Non-mechanical methods :

i. Decompression:

• In this method, large amount of N₂ gas is used to dissolve cell and high pressure (25000 Psi) is applied when gas pressure is suddenly released, N₂ gas comes out of cell forming gas bubbles and burst causing disruption of cell.
• Advantages:
  • No heat generation
  • This method protects enzymes and cell organelles.
• Disadvantages:
  • Cannot be used for yeast and plant cells.

ii. Osmotic shock:

• In this method, either hypotonic or hypertonic solution is used.
• The cell suspension is placed on either of the solution which create osmotic shock.
• Hypotonic solution:
  • Plasmolysis occur, the water enters the cytoplasm of cell and well burst.
• Hypertonic solution
  • Plasmolysis occur, the cell shrinks due to loss of water from cell.
• This is not always effective so it is used in combination with other process.

iii. Thermolysis (Heat shock protein):

• This method is very easy and economical for heat stable product.
• High heat inactivates cell by disrupting cell wall and release intracellular products.
• The effect of heat depends upon various factors such as pH, temperature, chelating agent, ionic strength, presence of enzymes (proteolytic and hydrolytic), time etc.
• Disadvantage:
  • Cannot be used for heat labile substances
  • Spore forming bacteria are also resistant to this method.

iv. Freeze-thaw:

• This is commonly used method to disrupt animal and bacterial cell.
• In this method, the cell suspension is frozen in dry ice an ethanol or freezer and then thaw the suspension at room temperature (37^oC).
• This suddenly freezing and thawing causes the cells to swell and break.
• The process is repeated for several time for efficient lysis.
• Disadvantages:
  • Very slow process
  • Not always effective

iv. Dessication:

• In this method, the cell suspension is dried by air drying or vacuum drying.
• After dessication, the cell shrinks.
• If excess water is provided than cell burst.
• This process is repeated for several time.
• Disadvantages:
  • Slower process and require several repeats.
  • Always used in combination with other process.

v. Electric lysis (Electroporation):

• When cell suspension is placed in electric field with intensity higher than certain threshold, there is formation of nano scale pore on cell surface.
• This pore maybe reversible or irreversible, depending upon applied field and direction of field.
• Through the pores, the intracellular material releases out.
• Disadvantages:
  • Heat generation
  • Expensive process

Chemical methods of cell disruption:

• Chemical permeabilizer
• Antibiotics
• Detergents
• Chartrops
• Chelating agents
• Hydroxides and hypochlorides

Biological methods or enzymatic methods of cell disruption

• Autolysis
• Lytic enzyme
• Phage lysis

Methods of Cell disruption: Cell lysis methods

The post Methods of Cell disruption: Cell lysis methods appeared first on Online Biology Notes.

• on the basis of International Union of Biochemistry (IUB) system:
  1. Oxidoreductase
  2. Transferase
  3. Hydrolase
  4. Lyase
  5. Isomerase
  6. Ligase

1. Oxidoreductase enzyme:

• The enzyme that catalyze transfer of H⁺ atom, or electron pair from one substrate to another is called oxidoreductase.
• According to International union of biochemistry and molecular biology (IUBMB), this enzyme belongs to class 1 and it is designated by E.C.1.
  e.g. E.C.1.1 – CHOH group containing donor
  E.C.1.1.1.- If NAD⁺ in NADP⁺ acts as electron acceptor.
• Examples of oxidoreductase enzyme:
  • Dehydrogenase, reductase, catalase etc.

2. Transferase enzyme:

• The enzyme that catalyze transfer of any group except H-atom such as methyl group, acetyl group, phosphate group etc. among substrate is called transferase.
• E.g. Acetyltransferase, kinase, methylase, polymerase, etc.
• This enzyme belongs to E.C.2
• Nomenclature:
  • E.C.2- transferase
  • E.C.2.1- enzyme that transfer one carbon group
  • E.C.2.1.1- if the group transfer is accepted by nicotinamide.

3. Hydrolase enzyme:

• The enzyme that catalyzes the break down of bond of substrate by using water is called hydrolase.
• The bond may be ester, glycosidic, peptide etc.
• E.g. digestive enzymes- pepsin, lipase, urease, proteinase, glycosidase
• This enzyme belongs to E.C.3
• Nomenclature:
  • E.C.3- Hydrolase
  • E.C.3.1.1- carboxylic ester hydrolase
  • E.C.3.1.1.3- Lipase (Glycerol ester hydrolase)

4. Lyase enzyme:

• The enzyme that catalyze the addition of removal of group from one substrate to another by mechanism other than hydrolysis and oxidation creating double bond is called lyases.
• E.g. aldolase, fumarase, histidase, carboxylase, ketolase etc.
• Nomenclature:
  • E.C.4 – lyase
  • E.C.4.1 – C-C lyase
  • E.C.4.1.1.1- Pyruvate decarboxylase
  • E.C.4.2.1.2- Fumarase

5. Isomerase enzyme:

• The enzyme that catalyze the interconversion of optical, geometrical, positional isomers by intermolecular re-arrangement of atom or group within substrate is called isomerase.
• It belongs to enzyme class E.C.5 e.g. Isomerase, Epimerase, Racemase
• Nomenclature:
  • E.C.5- Isomerase
  • E.C.5.1 – Epimerase and Racemase
  • E.C.5.1.1.1 – Alanine racemase
  • Glyceraldehyde-3-phosphate —-triosphosphate isomerase———-> Dihydroxyacetone phosphate

6. Ligase enzyme:

• The enzyme that catalyze the formation of bond such as C-C, C-N, C-S, C-O etc. utilizing the energy due to simultaneous breaking of pyrophosphate bond of ATP molecule or similar compound. E.g. ligase, synthetase
• Nomenclature:
  • E.C 6- ligase
  • E.C.6.1- form C-O bond
  • E.C.6.1.1 – aminoacyl tRNA synthase

Enzyme classification with examples

The post Enzyme classification with examples appeared first on Online Biology Notes.

• Enzyme inhibitors are the substance which when binds to the enzyme reversibly or irreversibly, decreases the activity of enzyme and the process is known as enzyme inhibition.
• Enzyme inhibitors are used to gain information about the shape of active site of enzyme and amino acids residues in active site.
• They are used to gain information about regulation or control of metabolic pathway.
• They can be used for drug designing.
• They are important for correcting metabolic imbalance.
• They are used for designing herbicides, pesticides and for killing pathogen.

Types of enzyme inhibitors:

I. On the basis of specificity:

II. On the basis of origin:

1. Natural enzyme inhibitor:
   • E.g. Alfatoxin, – amanitin
2. Artificial enzyme inhibitor (synthetic):
   • E.g. drugs

III. On the basis of whether the inhibition is reversible or irreversible

1. Reversible inhibition:

• The enzyme inhibition in which the enzymatic activity can be regained after removal of inhibitors.
• Types of reversible inhibition:
• i). Competitive inhibition
  • Competitive inhibitors are substrate analog that bind to substrate binding site of enzyme i.e. active site so competition occurs between inhibitor and substrate for binding to enzyme.
  • This type of inhibitor is overcome by increasing the concentration of substrate.
  • The kinetics of reaction is V_max remains same and K_m increases.
  • In this reaction, initially inhibitor binds to enzyme but with increase in concentration of substrate causes release of inhibitor.
  • Then, substrate bind enzymes so that the V_max remains same while K_m increases.
  • Example:
    • Succinate dehydrogenase convert succinate to fumarate.
      Succinate —succinate dehydrogenase————–> Fumarate + NADH +H⁺
    • Malate is competitive inhibitor of succinate due to structural analogy.
    • Malate + NAD⁺ —–succinate dehydrogenase———> Oxaloacetate
    • Sulphonamide is competitive inhibitor of PABA during tetrahydrofolate synthesis.
  • Example:
    • Treatment of methanol poisoning:
    • Methanol —–alcohol dehydrogenase————-> Formaldehyde (toxic)
    • Ethanol ——alcohol dehydrogenase———> Acetaldehyde
• ii). Non-competitive inhibition:
  • In this inhibition, there is no competition between substrate and inhibitor because the inhibitor binds to enzyme other than substrate binding site.
  • Since the binding site of substrate and inhibitor to enzyme is different, inhibitor don’t affect the affinity of enzyme to substrate.
  • In this case, the inhibition cannot be overcome by increasing substrate concentration.
  • The kinetic reaction is V_max decreases and K_m remains same. This means that substrate concentration has no effect on inhibition.
  • Binding of substrate and inhibitor are equal.
  • The inhibitor changes the conformation of enzyme after binding so that substrate cannot bind to enzyme.
  • This results in decrease of V_max.
  • Example:
    • Heavy metal poisoning. Hg, Pb etc. distort the -SH group containing enzyme at allosteric site.
    • Deoxycycline is non-competitive inhibitor of proteinase enzyme of bacteria.
    • The non-competitive inhibitor can be removed by pH treatment or by hydrolysis.
    • In case of metal poisoning, chelator is used.
• iii). Uncompetitive inhibitor:
  • This type of inhibition is seen in multi-substrate reaction.
  • It is rare type of inhibition.
  • The process of inhibition is same as non-competitive but it only binds to ES-complex.
  • At first substrate binds to enzyme to form ES-complex.
  • After binding of substrate to active site of enzyme, the binding site for inhibitor forms at allosteric site so that inhibitor bind.
  • The binding of inhibitor distorts the active as well as allosteric site of enzyme, inhibiting catalysis.
  • In this inhibition, V_max as well as K_m both decreases.
  • Examples:
    • Inhibition of lactate dehydrogenase by oxalate.
    • Inhibition of alkaline phosphatase by L-phenylalanine.
• iv. Mixed inhibition:
  • This type of inhibition is commonly seen in multi-substrate reaction.
  • It is the combination of competitive as well as non-competitive inhibition.
  • The mixed inhibitor can bind to both active site and allosteric site.
  • The kinetics of reaction is V_max decreases and K_m increases.
  • The V_max decreases because inhibitor non-competitively bind to allosteric site and distort enzyme.
  • Similarly, K_m increases because inhibitor can also bind to active site competiting with substrate.
  • This type of inhibition cannot be removed by increasing substrate concentration.
  • Examples:
    • Ketoconazole is mixed inhibitor bind to 5–α reductase enzyme.
    • Pallidium ion is mixed inhibitor of oxidoreductase enzyme.

2. Irreversible inhibition:

• In this type of inhibition, inhibitor bind to functional group of active sites by strong bond such as covalent bond and permanently destroy the catalytic property of enzyme.
• The functional group of active sites are -OH, -SH, -NH₂, etc.
• The irreversible inhibitor is non-specific and cause dead end of enzyme activity.
• The inhibitor can bind free enzyme and ES complex and destroy it permanently.
• Examples:
  • Iodoacetamide (CH₂ICOONH₂) bind with -SH group of enzymes permanently.
  • Enzyme-SH + CH₂ICOONH₂ —-> Enzyme-SCH₂COONH₂ + HI

Enzyme inhibition and types of enzyme inhibitors

The post Enzyme inhibition and types of enzyme inhibitors appeared first on Online Biology Notes.

• Purity is defined by the general level of protein contaminants and also by the absence of contaminants of special interests such as microbes, toxins etc.
• Protein purification is divided into five stages:
  1. Preparation of sources
  2. Knowledge of protein properties
  3. Development of an assay
  4. Primary isolation
  5. Final purification

I. Preparation of sources:

• The raw materials from which proteins can be isolated such as microbial culture or animals or plant sources should be selected.
• The amount of protein can be increased by increasing cultivation volume.

II. Knowledge of protein properties:

• Before employing any procedure, one should know about different properties of proteins such as- intracellular or extracellular occurrence, denaturation temperature, pH range, ionic stability, molecular weight, charge, iso-electric point, binding partners etc.

III. Development of an assay:

• An assay developed should be convenient, easy, rapid, precise for purification.

IV. Primary isolation:

• This consists of separation of protein from other cellular components.
• For this propose there are different methods:
• Concentration:
  • Different methods can be employed for concentration of extracellular protein.
  • Ultrafiltration are usually used to concentrate extracellular proteins from cell.
  • Ultrafiltration is a membrane filtration in which hydrostatic pressure is applied which causes movement of solution across the semi-permeable membrane.
  • Water and low molecular weight solute pass while other high molecular weight of molecules trapped in membrane.
  • The protein molecules are adsorbed in the membrane surface.
• Cell lysis: (For intracellular protein)
  • The intracellular proteins are liberated by cell lysis.
  • There are different methods for cell lysis.
  • They are:
  • Physical method:
  • Mechanical method: Bead mill, Homogenizer, Microfluidizer, Sonicator, French press/ X-mesh
  • Non-mechanical method: Decompression, Osmotic shock, Thermolysis, Freeze thaw, Dessication, Cell bomb
  • Chemical method:
  • Chemical permeabilizer whents
  • Antibiotics
  • Detergents
  • Chartrops
  • Chelating agents
  • Hydroxides and hypochlorides
  • Enzymatic method:
  • Autolysis
  • Lytic enzyme
  • Phage mediated lysis
• After cell lysis, the cellular constituents are concentrated by ultrafiltration.
• Refolding:
  • The first step in the refolding is the dissolution of the inclusion bodies (obtained from concentration) in a strong chaotropic solution of 6M urea, 2M thiourea.
  • Chaotropic agents are denaturating agent.
  • Chaotropic agents disrupt the intramolecular force between water molecules and allows protein and other macromolecule to dissolve easily.
  • The denaturated protein is then allowed to renature by removing the chaotropic agent by dilution, dialysis or by chromatographic separation.

V. Final purification:

• Chromatography is the usual method for obtaining pure protein.
• There are different types of chromatographic methods such as:
• Ion-exchange chromatography:
  • In case of ion exchange chromatography, cation or anion is attached to resin beads, depending upon the electric property of proteins.
  • If the desired protein is -vely charged then +ve charged resin beads are used.
  • The resin beads is packed in the column.
  • When the sample is poured in the column, the -vely charged protein (desired protein) stick on the beads while other undesired +vely charged protein eluted first.
  • The desired protein (-ve) is obtained as elute by changing the pH of the wash buffer or by washing with high salt solution.
• Hydrophobic chromatography:
  • This chromatography was developed to purify proteins by exploiting their surface hydrophobicity.
  • Groups of hydrophobic residues are scattered over the surface of proteins in such a way that it gives characteristic property to each protein.
  • The hydrophobic groups are covered by ordered layer of water in aqueous solution.
  • When salt is added then hydrophobic groups are exposed and interact with each other.
  • In hydrophobic interaction chromatography, the column is packed with hydrophobic beads (-phenyl, -acetyl group).
  • When the sample is poured, the hydrophobic protein interacts with hydrophobic matrix.
  • The salting-out compound such as ammonium sulfate is used from high to low concentration in the column so the protein with low hydrophobicity elute first.
  • The non-ionic detergents such as tween-20, triton-x-100 etc. are used to elute the protein.
• Affinity chromatography:
  • In affinity chromatography, a compound having specific affinity to desired protein is attached to the resin. For. e.g. Antibody against desired protein is coated on resin.
  • The resin is then packed into a column. When mixture of protein is poured, only those proteins having specific affinity with resin (Ab coated) stick on the column.
  • All the other protein gets eluted.
  • Only the undesired protein gets eluted.
  • The protein of interest stuck on the column can be eluted by changing the ionic strength of the solution so that the desired protein no longer binds to resin and get eluted.
  • This can also be achieved by adding special compound on elution solution which change the equation state and elute the protein.
• Size-exclusion chromatography:
  • This process is also known as gel filtration.
  • The method used to separate proteins on the basis of their size or molecular weight.
  • The porous matrix is packed in the column.
  • The porous matrix retards the rate of elution of proteins.
  • The protein with higher molecular weight elutes first since small protein passes through pores and elute last.

Protein characterization:

• The methods of protein characterization are:

i. Electrophoresis:

• It is the process of separation of charged particles under the influence of electric field.
• SDS-PAGE is the most widely used method for analysis of protein in the mixture.
• It is useful for monitoring the protein purification.
• SDS-PAGE separates proteins on the basis of molecular weight.
• At first polyacrylamide gel is made and a well is made.
• The protein sample is mixed with beta-mercaptoethane and sodium dodecyl sulphate (SDS) and boiled for 5 minutes.
• During boiling, proteins get denatured.
• Each SDS molecule binds to two amino-acids molecules of denatured protein.
• SDS molecule is highly -vely charged so the protein binds with SDS become -vely charged.
• When electrophoresis is done, the protein moves towards anode (+ve charge).
• The small size protein migrate faster and large size moves slower forming different band.
• The band can be visualized by staining with Coomassie brilliant blue (CBB).

ii. Peptide sequencing:

• This method is developed by Pehr Edman so it is also known as Edman degradation.
• The polypeptide is reacted with phenylisothiocyanate under mild alkaline condition.
• The amino terminal of peptide is converted to phenylthiocarbomyl (PTC).
• The phenylthiocarbomyl (PTC) derivatives is washed thoroughly with organic solvent (e.g. benzene) and dried.
• The dried PTC is treated with anhydrous acid (e.g. heptafluorobutyric acid).
• This results in cleavage of PTC-polypeptide near PTC substitution releasing N-terminal aminoacid as thioazoline derivatives.
• The thioazoline derivative is stable. So, it is converted to thiohydantoin derivative containing aminoacid is identified by high performance liquid chromatography (HPLC).
• If the aminoacid is alanine then the first aminoacid is the polypeptide along N-terminal is alanine.
• The Edman degradation process is repeated for sequencing other amino-acids.

iii. Tryptic mapping:

• Edman degradation method for determining amino-acid sequence from N-terminal require free amino-group at N-terminal of protein.
• However, 50-70% of proteins have N-terminal blocked by Formyl, acetyl or acryl group during post translational modification.
• For such protein, sequencing is not possible so, the protein have been cleaved by endopeptidases to produce peptide which is then sequenced.
• Trypsin enzyme is an example of endopeptidases which cleaves C-terminal of arginine and lysine.
• Similarly, other endopeptidases have their own restricted cleavage site.
• The generated short fragment is sequenced by Edman method followed by HPLC to identify the aminoacids.

iv. Analytical ultracentrifugation:

• This method measures variety of properties of protein sample including molecular weight, interaction with other molecules and sample homogeneity.

v. Spectroscopy:

• It is used in analysis of wide range of sample.
• The metal containing protein (co-factors) can be analysed by spectroscopy.
• The different co-factor gives different electromagnetic spectrum.

vi. Biosensors:

• It is a device used for the detection of particular protein in cell.
• The device is coated with specific Ab against the desired protein.
• When sample is added, the particular protein binds the Ab producing signal on device.

vii. Mass spectroscopy:

• Mass spectroscopy is an analytical technique that provide information about molecular structure of organic and inorganic compound.
• The mass of particular protein can be determined by mass spectroscopy.
• It can detect post translational modification or any variation in structure or protein.

Protein purification and characterization

The post Protein purification and characterization appeared first on Online Biology Notes.

• In the technique of mass spectrometry, the compound under investigation is bombarded with a beam of electron which produces an ionic molecule or ionic fragments of the original species.
• Then, the separation of the resulting assortment of charged particles is done according to their masses.
• The spectrum produced, known as mass spectrum is a record of information regarding various masses produced and their relative abundances.
• Mass spectrum is an analytical technique which can provide information concerning the molecular structure of organic and inorganic compounds. It can be used to determine directly molecular weight as high as 4000.
• It is one of the few methods that can be used as a qualitative analytical tool to characterize different organic substances.
• With it, one can do analysis of mixture (gases, or liquids, and in some cases solids) quantitatively.
• A mass spectrometer is also useful to investigate reaction mixtures and in tracer work.
• It is also used in understanding kinetics and mechanisms of unimolecular decomposition reaction.
• Although the mass spectrometer is based on simple principle, yet it is a very complex and very expensive instrument.
• Nevertheless, it is becoming a common tool because it gives the largest amount of specific information about the substances to be analysed.

Principle of Mass Spectrometer:

• The mass spectrometer is an instrument which helps in separating the individual atoms or molecules because of the difference in their masses.
• Let us consider, a molecule M, which is bombarded with a beam of electrons.
• Suppose this is ionized as follows:
  • M +e^–à M⁺+ 2e^–
• Where M⁺ is an ionized molecule and e^– is an electron. The ions are now accelerated in an electric field at voltage V.
• If this is the condition, the energy given to each particle is eV and this is equal to the kinetic energy which is equal to ½ mv².
• This can be expressed as:
  • ½ mv²=eV———————– equation (1)
  • Or, v²= 2eV/m
  • Or, v= m—————-equation (2)
• Where v is the velocity of the particle of mass m, e is the charge on an electron and V is the accelerating voltage.
• The same energy eV is possessed by all the particles.
• Also, all particles have the same kinetic energy ½ mv². As the value of m varies from the particle to particle, the velocity v also changes such that ½ mv² remains a constant.
• For a particle of mass m₁ and velocity v₁, equation 1 becomes as:
  • ½ m₁v₁²=eV———————– equation (3)
• Similarly, for a particle of mass m₂ and velocity v₂, equation becomes as:
  • ½ m₂v₂²=eV———————– equation (4)
• And for particles m₃, m₄……. Of velocities v_3, v₄…..respectively, the equation (1) becomes as:
• ½ m₃v₃²=eV———————– equation (5)
• ½ m₄v₄²=eV———————– equation (6)
• ½ m₅v₅²=eV———————– equation (7)
• From equation 3,4,5,6 and 7, we have
• ½ m₁v₁²= ½ m₂v₂²= ½ m₃v₃²= …….. and so on….. equation (8)
• From relation (8), it shows that the velocity of different particles will vary, depending on the mass of the particles.
• After the charged particles have been accelerated by an applied voltage, they enter a magnetic field H.
• This field attracts the particles and they move in a circle around it.
• This attractive force, due to magnet is HeV, whereas the balancing centrifugal force of the particle is mv²/r.
• As the particle starts moving uniformly around the circular path, the two forces become equal, i.e.
  • mv²/r= HeV
  • Or, 1/r = HeV/mv²
  • Or, r = mv/eH ————-equation (9)
  • Where ‘r’, is the radius of the circular path of the particle
• On substitution equation (9) in (2), we get
  • r= meH
• On squaring both sides,
  • r²= m²/e²H² 2eV/m ————equation (10)
  • Or, r = ²e) ————-equation (11)
• Equation (10) can be written as m/e= H²r²/ 2V———equation (12)
• From equation (11), it follows that the radius of the circular path of particles depends on the accelerating voltage V, the magnetic field H, and the ratio, m/e.
• As e, V and H are constant, it means that the radius of the ionized molecule depends on m, its mass.
• The relation between m, the radius of the circular path of the ion, V and H i.e., equation (11) is the basis of separation of particles according to their masses.
• Thus, the radius of the path may be changed by varying either the magnetic field (H) or the accelerating voltage (V).
• By, either method, ions of the different mass to charge ratio (m/e) can be made to impinge upon the collector in turn thus giving rise to a spectrum.

Instrumentation of mass spectrometer:

• The mass spectrometers used for the investigation of any compound may vary in their types but generally all contain the following components:
• The inlet system (or sample handling system):
  • As a mass spectrometer should have a vapor sample and to ensure that the sample enters the ionization chamber at a constant rate, the sample is converted into the gaseous state in the inlet system.
  • To achieve this, the system is usually heated.
  • To handle different types of materials, different inlet systems are employed.
  • In order to transfer gases, these involve merely transfer of the sample from a gas bulb into the metering reservoir and then expand into an expansion reservoir, having volume of three liters.
  • In the metering reservoir, the pressure ranges from 30-50 torr and after expansion the pressure ranges from 10^-3 to 10^-1 torr in expansion reservoir.
  • Liquid samples are handled by hypodermic needle injection through silicone rubber dam.
  • As the reservoir has low pressure, it draws the liquid immediately and vaporizes it instantly.
  • From the sample reservoir, the gaseous samples are leaked into the ionization chamber or ion source through a pinhole restriction of about 0.013 to 0.050mm in diameter in a gold foil.
  • For MS, a sample size of about 1 µmole is required.
• The ion source or ionization chamber:
  • From the inlet system, the sample is introduced into the ionization chamber where the beam of the electrons is put across the molecules of the samples.
  • The molecules become ionized. The electron beam is accelerated by a high voltage up to 100V on the collector, and then the collision between electrons and molecules results in the production of well-defined fragments, carrying a definitive positive charge.
  • The electrostatic accelerating system: The positive ions formed in the ionization chamber are withdrawn by the electric field.
  • A strong electrostatic field of 400-4000V accelerates the ions of masses m₁, m_2, m₃…to their final velocities.
  • Whenever the mass spectrometer is started to record the spectrum, the second accelerator is charged to an initial potential of 4000volts.
  • Then, this charge is permitted to leak off to ground at a controlled rate over a period of 25minutes.
• Magnetic field:
  • As the accelerated particles from the electrical field enter the magnetic field, the force of the magnetic field requires of them to move in a curved path.
  • The radius of this curvature, r , relies upon the mass m, the accelerating voltage V, the electron charge, e, and the strength of the magnetic field, H.
  • It is the two properties m/e and r upon which mass spectrometry is based.
  • The mass to charge ratio and the radius to the curvature are interdependent, whereas a change in either the accelerating potential or the magnetic field will change m/e and r.
  • In the magnetic deflection, mass spectrometer the radius, r is fixed and all the particles of a single mass to charge ration m/e, are focused on the detector at any given applied potential.
• The ion separator (analyzer):
  • This is that part of MS which separates ions according to their masses.
  • An analyzer must possess the following characteristics:
  • It should have a high resolution.
  • It must have a high rate of transmission of ions.
• The ion collectors:
  • The ion beam currents are of the order of 10-15 to 10-19 ampere.
  • This small current has to be detected in mass spectrometers.
  • Photographic plates, Faraday cylinders, electron multipliers and electrometers are generally employed.
  • The readout display usually possesses a direct writing recording oscillograph which has 3-5 galvanometers with relative sensitivities of 1, 3, 10, 30 and 100.
  • This method is cheap, easy to operate and reliable.
• Vacuum system:
  • A high vacuum is to be maintained in the instrument.
  • The inlet system is generally maintained at 0.015 torr, the ion source at 10-3 torr and analyzer tube at 10-7 torr or as low as possible.
  • The oil diffusion and mercury diffusion and mercury diffusion pumps are commonly used in different types of combinations.

Applications of mass spectrometer:

• For Molecular mass determination
  • The isotopic abundance of easily vaporizable elements can be determined
  • Mass spectrometry is used to determine the amount of component of a complicated mixture from which it cannot be separated quantitatively. This determination is often made possible by the determination of isotope ratios.
• For Quantitative analysis of mixtures
  • Mass spectrometry is successful in making distinction between cis- and trans- isomers.
• For Evaluation of heat of sublimation
  • MS spectrometer can be used to determine the bond-dissociation energies of molecules by employing the concept of appearance potential of a fragment ion.
  • MS is very frequently used in the kinetic and mechanistic reaction studies.
  • MS can be used for the determination of latent heat of vaporization of liquid using the Clausius-Clapeyron equation which expresses the saturated vapor pressure of a liquid as a function of the absolute temperature.
• MS is one of the best methods to detect impurities.
• MS is used for characterization of polymers.

Mass spectrometry Principle, instrumentation and applications

The post Mass spectrometry Principle, instrumentation and applications appeared first on Online Biology Notes.

• The term electrophoresis describes the migration of a charged particles under the influence of an electric field.
• Various essential biological molecules, such as amino acids, peptides, proteins, nucleic acids, nucleotides, have ionizable group, which at given pH exist in a solution as electrically charged species either as cation (+ve) and anion (-ve) are separated by electrophoresis
• Under the influence of electric field these charged particles will migrates either to cathode or anode depending on the nature of their net charge

Principle of electrophoresis:

• When a potential difference is applied, the molecules with different overall charge will begin to separate owing to their different electrophoretic mobility. Even the molecules with similar charge will begins to separate if they have different molecular sizes, since they will experience different frictional forces. Therefore, some form of electrophoresis rely almost totally on the different charges on the molecules for separation while some other form exploits difference in size (molecular size) of molecules.
• Electrophoresis is regarded as incomplete form of electrolysis because the electric field is removed before the molecules in the samples reaches the electrode but the molecules will have been already separated according to their electrophoretic mobilities.
• The separated samples are then located by staining with an appropriate dye or by autoradiography, if the sample is radiolabeled.

Factor affecting electrophoresis:

Instrumentation of electrophoresis:

• Equipment required for electrophoresis basically consists of two items, a power pack and an electrophoresis unit.
• Electrophoresis units are available for running either vertical or horizontal gel systems.
• Vertical slab gel units are commercially available and routinely used to separate proteins in acrylamide gels.
• The gel is formed between two glass plates, clamped together but held apart by plastic spacers.
• Gel dimensions are typically 12cmX 14cm, with a thickness of 0.5 to 1mm.
• A plastic comb is placed in the gel solution and is removed after polymerization to provide loading wells for samples.
• When the apparatus is assembled, the lower electrophoresis, tank buffer surrounds the gel plates and affords some cooling of the gel plates.
• The gel is cast on a glass or plastic plates and placed on a cooling plate (an insulated surface through which cooling water is passed to conduct away generated heat.)
• Connection between the gel and electrode buffer is made using a thick wad of wetted filter paper, however the agarose gels for DNA electrophoresis are run submerged in the buffer.
• The powerpack supplies a direct current between the electrodes in the electrophoresis unit.
• All electrophoresis is carried out in an appropriate buffer, which is essential to maintain a constant state of ionization of the molecules being separated.
• Any variation in pH would alter the overall charge and hence the mobilities (rate of migration in the applied field) of the molecules being separated.

Different types of electrophoresis:

Electrophoresis Principle, affecting factors and types

The post Electrophoresis Principle, affecting factors and types appeared first on Online Biology Notes.

• A large number of substances are unknown which can absorb ultraviolet or visible light energy.
• But these substances lose excess energy through heat through collisions with neighboring atoms or molecules.
• However, a number of essential substances are also known which lose only part of this excess energy in the form of heat and release the remnant energy as electromagnetic radiation of a wavelength longer than that absorbed.
• The process of emitting radiation is collectively known as luminescence.
• In luminescence, light is produced at low temperatures.
• Thus, the light emitted by this process is regarded as ‘light without heat’ or ‘cold light’.
• Luminescence is of 2 types:
• i.          Fluorescence:
  • When a beam of light is incident on certain materials, they emit visible light or radiations.
  • This phenomenon is known as fluorescence and the substance showing this phenomenon is known as fluorescent substances.
  • The phenomenon of fluorescence is instantaneous and starts immediately after the absorption of light and stops as soon as the incident light is cut off.
• ii.         Phosphorescence:
  • When light radiation is incident on certain materials, they continue to emit light even after the incident light is cut off.
  • This type of delayed fluorescence is called phosphorescence and the substances are called phosphorescent substances.
  • A material exhibiting fluorescence generally re-emits excess radiation within 10-6 to 10-4 seconds of absorption.
  • On the other hand, materials exhibiting phosphorescence re-emit excess radiation within 10-4 to 20 seconds or longer.

Principle of Fluorimetry:

• When molecules are irradiated with light of the appropriate frequency, it will be absorbed in about 10-15 seconds.
• In the process of absorption, the molecules may move from ground to the first excited singlet electronic state.
• Although at room temperature molecules may be present in their ground vibration level.
• After absorption, the excitation molecules can end up in any one of the vibrational levels in the first excited electronic state.
• From the excited singlet state, one of the following three phenomena will probably occur, depending on the molecule involved and the conditions:
• The first possibility is that the excited singlet state is relatively unstable, in such a situation, the excited molecules will return to the ground state by collisional deactivation without emitting any radiation.
• The second possibility is that the molecules in the excited singlet state may emit an ultraviolet or visible light photon. This process is known as fluorescence.
• The third possibility is that the molecule with a relatively stable excited state may undergo transition and sometime thereafter returns to the ground state, usually by the emission of an ultraviolet or visible light photon. This is known as phosphorescence emission.
• The instruments used for the measurement of fluorescence are known as fluorimeters.
• In these, filters are used to isolate the wavelength of excitation.
• Thus, a fluorimeter is a manual instrument and is best used for measurements at one or two wavelengths because a change in filters is to be made each time the wavelength is changed.
• A fluorimeter employs a mercury vapor lamp, a condensing lens, a primary filter, a sample container, a secondary filter, and a receiving photocell.
• Generally, the primary filter is used to select ultraviolet but not visible radiation whereas the secondary filter is used to transmit visible fluorescent radiation and to absorb incident ultraviolet radiation.
• The light from the mercury vapor lamp is allowed to pass through the condensing lens followed by its passage through a primary filter.
• The primary filter selects only UV radiation but absorbs visible radiation.
• The UV radiation from the primary filter is passed through a sample container.
• From the sample, UV and fluorescent radiations are obtained which are passed through a secondary filter that absorbs the primary radiant energy but transmits the fluorescent radiation.
• This is received by a photocell placed in a position at right angles to the incident beam.
• The output of the photocell is measured by a sensitive galvanometer or another device.

Applications of Fluorimetry:

• Determination of uranium in salts used extensively in the field of nuclear research.
• Estimation of traces of boron in steel by means of the complex formed with benzene.
• Estimation of calcium by fluorimetry with a calcium solution.
• Determination of Vitamin B (B1 thiamine and B2 riboflavin) in the food samples like meat, cereals, etc.
• Fluorimetry is employed to carry out both qualitative and quantitative analyses for various aromatic compounds present in cigarette smoke, air-pollutant, concentrates, and automobiles exhaust.

Fluorimetry-Principle and Applications

The post Fluorimetry-Principle and Applications appeared first on Online Biology Notes.