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DETERMINATION OF MOLECULAR WEIGHT OF MACROMOLECULES WITH REFERENCE TO PROTEIN AND DNA:

Macromollecules are very large molecules such as proteins and DNA. Molecular weight of a molecule .Is the sum of the relative atomic masses of all the atoms of that molecule. DETERNINATION METHODS 1. BY SEDIMENTATION METHOD, Sedimentation is a biochemistry method that provides hydrodynamic and thermodynamic information about the purity, size, shape molar masses ,association energy, association stoichiometry, and thermodynamic non ideality of molecules in solution. The fundamental measurement in sedimentation is the the concentration as a function of radial position. Any of the three profiles provides the necessary concentration hence a useful tool in biological solutions analysis. Sedimentation describes the motion of molecules in solution and particles in suspensions in response to an external force such as gravity. Sedimentation involves settling of solid particles through a liquid either to produce a concentrated slurry from a dilute suspension or to clarify whether a liquid contains solid particles. When particles are forced through a solution, they experience resistance to movement which depend s on the properties of the particle such as mass, shape , and density and properties of the solvent such as its temperature , viscosity , density and composition. A commonly used experimental format where it occurs is sedimentation in a centrifugal field which is also called Centrifugation. This is used as a preparative strategy to separate complex mixtures present in biological sample. They are two methods of sedimentation and these are 1. Sedimentation velocity 2. Sedimentation equilibrium. Data analysis from both methods use computer programs developed around fundamental equations such Lamn equation,Svedbverg equation. Sedimentation velocity .is used to check for impurities and molecular interactions . Sedimentation equilibrium is used to provide first principle insight into molecular interactions such as macromolecular binding and thermodynamic nonideality.

Physical basis of centrifugation :Centrifuge instrumentation.

The centrifuge is a high speed device in which solid or liquid particles of different densities are separated by rotating them in a tube in a horizontal circle. The denser particles tend to move along the length of the tube to a greater radius of rotation, displacing the lighter particles to the other end. Its electrically driven.
Analytical ultracentrifuge.

In analytical ultracentrifuge,

Sample is placed in a sample cell in a rotor contained in an evacuated and refrigerated chamber. Centrifugation is carried out at very high speed during which the behaviour of the sample is analysed. The cell is scanned with parallel light as it passes through the detection system on each revolution. Transparent windows at the top and bottom of sample cells allow light to pass through the sample. Optical systems use absorbance, fluorescence or refractive index to detect sample concentration as a function of position in the cell. Refractive index measurements of sample is continuously compared to that of solvent alone in a double-sector sample cell shown on the right. This data is continuously collected during the experiment, stored in amicrocomputer and subsequently analyzed.

Sedimentation velocity : Is an analyticalultracentrifugation (AUG ) method that measures the rate at which molecules move in response to a centrifugal force generated in a centrifuge.this

sedimentation rate provides information about both the molecular mass and shape of the technique can also measure diffusion coefficients.
Operation

Sedimentation velocity analysis:- when the sample is spun at a very high speed usually
40 60 kpm in an analytical ultracentrifuge. It Involves following the behavior of sample particles as they move through the solvent during sedimentation at relatively high centrifugal speeds. Particles become distributed in a concentration gradient of characteristic shape which changes during the experiment . At the beginning of the experiment, sample is distributed uniformly through the sample cell. The lamella of molecules at the interface between this solution and air is referred to as the boundary. During centrifugation, these molecules move towards the bottom of the sample cell which causes a change in the boundary position and shape which may be followed by absorbance, fluorescence or refractive index. This is because sedimentation forces the sample particles to move through the solvent away from the centre of rotation while diffusion causes spreading of the boundary resulting in a change in boundary shape during the experiment. Computer methods for analysis of these boundaries have been put in place using mathematical models making a number of physical and chemical parameters accessible to determination by this
method.

These include :-

1.The buoyant mass [mo(1 )],


2.Sedimentation and diffusion coefficients of individual molecular species. The sedimentation coefficient, S, is the ratio of velocity to acceleration of the centrifuged particle which is related to physical and hydrodynamic properties of the sample and solvent .

TREAMENT OF RESULTS Measuring the position, r , of a boundary formed by a dilute solution of the particle at any time, t, we could experimentally determine the apparent sedimentation coefficient, S, by sedimentation velocity. This is determined by measuring the rate of movement of the boundary through the column of solvent in the centrifugation cell;

S = M (1 V2 P ) / ( No 6 R ) Where
S is sedimentation coefficient. M is the mass of the protein molecule in Dalton; No is Avogadrosnumber, 6.0231023; v2 is the partial specific volume of the protein; typical value is 0.73 cm3/g; is the density of solvent (1.0 g/cm3 for H2O); is the viscosity of the solvent (0.01 g/cms for H2O). f is friction coefficient and is = 6 R A critical factor in the equation is the frictional coefficient, f (dimensions gram per second) which depends on both the size and shape of the protein.

In the sedimentation velocity method, the sample is spun at a very high speed usually 40 60 kpm in an analytical ultracentrifuge. The high centrifuge force rapidly depletes all the protein from regions nearest the center of the rotor, the meniscus region at the air solution interface, forming the sharp boundary between the solvent that has cleared of the particles and that still containing the sedimentation particle which moves towards the outside of the rotor with time, until all the sedimentation particles form a pellet Outside the cell. The concentration distribution across the cell at various times during the experiment is measured while the sample is spinning using either absorbence or refractive index detection. This method enables the sample analysis at various conditions such as temperature, pH, ionic strength , The amount of the sample depends on applications but usually its between 0.45 mL at concentration of 0.1 mg/mL ( 45 450 ) mg

A known volume of the sample is placed in a sample cell in a rotor contained in a evacuated and refrigerated chamber. Centrifuge is done at a very high speed around a central point to develop significant centrifugal force , during which the behavior o the sample is analyzed. The sample is centrifuged until the particles are tightly packed into a pallet at the bottom of the tube.The upper portion of the liquid is then separated by decantation . The fractions obtained can be assayed by radioactivity, chemical tests, enzymatic activity or acombination o the three. The sedimentation fractions can also be annalysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the standard sedimentation coefficient . The sedimentation coefficient of a protein is a measure of how fast it moves through the gradient. Increasing the mass of the protein will increase its sedimentation, while increasing its size or asymmetry will decrease its sedimentation.

Physical Basis of centrifugation.

Consider a particle of mass m, suspended in a solvent of density p,This paricle would experience an upthrust equivalent to the weight of displaced liquid in accordance to Archimeds princimple.The buoyant mass of the particle is therefore m minus the correlation factor of this upthrust.

Centrifugal force. A particle of mass, m, experiences a centrifugal force, F, when centrifuged at an angular velocity, ,around a point from which it is distant by a radius, r.
PRINCIMPLE

A particle , whether it is a precipitate ,a macromolecule, or cell organelle, is subjected to a centrifugal force when it is rotated at a high rate of speed, The centrifugal force is defined by F = M w2 r When sedimentation and diffusion come to a state of equilibrium, no apparent movement of solutes occurs hence sedimentation velocity is attained. The equilibrium concentration is dependant on the buoyant molecular weight, M ( 1- VP ). In order to determine the molecular weight of the the protein , accurate partial specific volume and density must be obtained. The relationship of Sedimentation coefficient S, to size and shape of the protein is given by the Svedberg formula:

S = M ( 1- V2 P ) / Nof

S = M (1 V2 P ) / ( No 6 R )
S is sedimentation coefficient. M is the mass of the protein molecule in Dalton; No is Avogadrosnumber, 6.0231023; v2 is the partial specific volume of the protein; typical value is 0.73 cm3/g; is the density of solvent (1.0 g/cm3 for H2O); is the viscosity of the solvent (0.01 g/cms for H2O). f is friction coefficient and is = 6 R A critical factor in the equation is the frictional coefficient, f (dimensions gram per second) which depends on both the size and shape of the protein.

OR PRINCIMPLE A particle , whether it is a precipitate ,a macromolecule, or cell organelle, is subjected to a centrifugal force when it is rotated at a high rate of speed, The centrifugal force is defined by

The relationship of Sedimentation coefficient S, to size and shape of the protein is given by the Svedberg formula: Principle :

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