Lecture 17: Application of Sedimentation concept and Epilogue Review Diffusion o Collisions amongst molecules o Model of random Walk o Friction and it’s relation to molecular structure o Fick’s laws o Sedimentation Today o Sedimentation in centrifuge o Density gradient centrifugation (application to DNA problems) o Concept of viscosity o Electrophoresis o Gel electrophoresis o DNA sequence analysis o Pulsed field gradient electrophoresis o Application to DNA and Proteins Density Gradient Centrifugation Using salt or sucrose solution where it’s difficult to overcome effects due diffusion it is possible to create density gradient across a tube in a centrifuge. Since sedimentation coefficient depends on: s (1 v) and (x)=top+(d/dx)x, it is possible to find a location where =1. At this location forces due buoyancy and centrifugation exactly cancel leading to zero mobility. This is where the molecular migration stops and can be used to achieve separation as shown below + Viscosity Consider laminar flow, generated in figure below. Liquid in contact with moving wall will move with velocity of the wall while the liquid at the bottom wall will be stationary. This creates a momentum gradient along y. The consequence of the momentum gradient is that there is net momentum flux transferred in the opposite direction. Each of these laminar layers creates an effective friction amongst layer whose magnitude depends on the pressure gradient along the x direction. The viscosity is therefore defined as, J mu du y dy Note that the frictional coefficient f is directly proportional to the viscosity coefficient. Viscosity (Continued) To measure viscosity coefficient, we can determine the terminal velocity of sphere and relate it to the viscosity of the fluid. Generally, same sphere can be used to compare viscosities of different fluids. However, more commonly an Oswald viscometer is used. Here a fixed volume of liquid is allowed to run through a glass capillary and time required is measured. The viscosity of the fluid is calculated by comparing it to the fluid of known viscosity. Measured viscosity also depends on the density of the fluid, since the pressure difference is directly related to it. 1 1t1 . This method is generally 2 1t1 suitable for reasonably low viscosity fluids. However, for a highly viscous fluid, a cone viscometer is commonly used. In this method, a spindle is immersed in viscous fluid and the force required to maintain certain rotational frequency is measured. This allows measurement of the viscosity as a function of rotational speed. Generally, polymer solutions show a dramatic dependence on the rotational speed. Hence they are termed as non-Newtonian fluids. By measuring the viscosity as a function of concentration one can attempt to relate the structural changes occurring under dynamic conditions. Gel-electrophoresis Just as in sedimentation processes, we exploited the balancing of forces involving gravity and friction. It is possible to extend this idea where we replace mechanical forces with electrical forces. In this way, molecules that have charge can be imparted with different terminal velocity, ZeE f The quantify u/E is termed as an electrophoretic mobility and the method is called as electrophoresis. Since DNA molecules have negatively charged PO4 groups, single strands of DNA molecules were sequenced using a clever technique. ZeE fu u Gel Electrophoresis Actually, most of the electrophoresis studies use gel media as opposed to solutions. Gels can be made with special polymers such as gelatin, agar, or polyacrylamide. The common features of gel that makes them valuable for these studies are: 1. Convection or accidental mixing is avoided. 2. Owing to their micro-porous structure they slow the speed of migration significantly depending on the size of the protein or DNA. 3. Polymer-bio-molecule interactions can be influenced by selecting the size of the network mesh (concentration) and/or charges on the gel forming polymer 4. Owing to the obstructive nature of the of polymer, the actual path taken by bio-molecules is much longer than the length of the gel allowing for better separation. (Think about the resolution obtained on a chromatographic column) As shown before, one of the clever methods to sequence DNA in seventies was to subject single stranded DNA to specific enzymes that cleave a specific base in DNA. As there are only 4 bases, this method allowed structural sequence of DNA to be determined using radio-labeled DNA. The trick here was to use 7m urea. It disables the base pairing interactions and leaves the charged phosphate groups unaffected. So the DNA migrates electrophoretically. To improve resolution and extend the range of the technique to higher molecular weights, a pulsed field gradient electrophoresis was invented. Applications to Proteins Fundamentally, the same ideas can be used to separate and identify new proteins. The frictional coefficients of the proteins depend on their size and shape. Also charge on the proteins is dependent on their basic amino acid sequence. The net charge depends on the PK and therefore on the pH of the buffer solution. Since different proteins have different isoelectric points (i.e. where the net charge on the protein is zero), a method of isoelectric focusing has been developed. Different buffers are used to establish a pH gradient in a gel. So, when a protein enters a region of pH corresponding to its isoelectric point, its mobility vanishes allowing for separation based on the net charge on the protein. Conceptually, this is similar to the density gradient method employed in sedimentation. Pulsed field Gradient Electrophoresis Protein Molecular weight Unlike DNA, which has fixed charge per base pair due to phosphate group, proteins can have variable charges depending on the amino acid configuration. To create a uniform charge density, proteins are denatured and treated with Sodium dodecyl sulphate and mercapto-ethanol. The latter cleaves the S-S thiol bonds. The former, when used in concentrations of 1 mM or above, binds strongly to the denatured protein (1SDS per two amino acid groups) leading to a uniform charge density per unit length. This SDS-PAGE method allows one determine molecular weight of the proteins based on their electrophoretic mobility as shown above.