Vithaldas P. Shanbhag

Affinity partitioning of proteins in aqueous two-phases systems containing polymer-bound fatty acids : I. Effect of polyethylene glycol palmitate on the partition of human serum albumin and α-lactalbumin

Abstract The partition of serum albumin between the two aqueous phases of a polyethylene glycol—dextran—water two-phase system is strongly influenced by replacing a small fraction of the polyethylene glycol by its palmitate ester. The partition of albumin is compared with the partition of polymer-bound palmitate using polyethylene glycol[1-14C]palmitate. The data indicate that a maximum (“saturation”) effect on the partition of albumin corresponds to a binding of fewer than two palmitate groups per albumin molecule The effect on α-lactalbumin corresponds to the maximum binding of 0.5 palmitate group per protein molecule. These data do not fit the most favoured available model for affinity partitioning. The deviation can be attributed to the observation that the palmitate groups probably form micelle-type aggregates in the polyethylene glycol-rich phase at the concentrations used for albumin extraction.

Affinity partitioning of biopolymers and membranes in ficoll-dextran aqueous two-phase systems

Abstract The usefulness of an aqueous two-phase system composed of water and the two polymers dextran and Ficoll for affinity partitioning of proteins, nucleic acids, synaptic membranes, and the thylakoid membrane system of chloroplasts was studied. In contrast to the widely used dextran—poly(ethylene glycol) two-phase system the two phases of the present system have very similar properties. The partition coefficients of proteins are close to unity. If polymer-bound ligands are introduced into one phase, ligand-binding biopolymers and particles can be extracted into that phase. Ligands, including fatty acids, diamines, triazine dyes and pharmaceutical substances (with affinity for opiate receptors), have been bound to dextran, Ficoll or poly(ethylene glycol). When used in small amounts, poly(ethylene glycol) partitions into the Ficoll-rich phase. The solubility of proteins in these two-phase systems is higher than in dextran—poly(ethylene glycol) systems, and also the tendency for membranes and cell particles to accumulate at the interface is markedly reduced.

Isoelectric points of membrane surfaces of three spinach chloroplast classes determined by cross-partition

Abstract The isoelectric points of the membranes surrounding three classes of spinach chloroplasts have been determined by partition at different pH values in aqueous two-phase systems where the electrical potential differences at the interface are opposite (cross-partition). Class I chloroplasts, intact chloroplasts, have an isoelectric point at pH 3.8–4.1 and class II chloroplasts, broken chloroplasts or intact thylakoid membranes, have an isoelectric point at pH 4.7–4.9. The third class of particles, class III ‘chloroplasts’, that contain one or more chloroplasts, mitochondria, peroxisomes and some cytoplasm all surrounded by a membrane, probably the plasma membrane, have an isoelectric point at pH 3.4–4.0. The partition technique used presumably yields the isoelectric point of the surface of the membranes exposed to the phase system by the three classes of chloroplasts, i.e., the outer envelope membrane, the thylakoid membrane and the plasma membrane, respectively. The isoelectric points obtained with this technique are suggested to reflect protein to charged-lipid differences in the composition of the membranes.

Calmodulin binding to human spectrin

Calmodulin is shown to interact with human spectrin dimer. The binding was highly calcium‐dependent and observed in two different kinds of experiments. Firstly, affinity chromatography of calmodulin on a Sepharose 4B column with immobilized spectrin, and secondly, partition in aqueous two‐phase polymer systems. In the column experiments stoichiometric amounts of calmodulin were retained on the spectrin‐Sepharose column when micromolar concentrations of calcium were present. The calmodulin bound could be eluted with EGTA. The partition coefficient of calmodulin in an aqueous two‐phase polymer system containing calcium was changed upon addition of spectrin, indicating an association between the two proteins. In the absence of calcium, spectrin did not cause any change in the partition behaviour of calmodulin, thus showing that the association requires calcium.

Diffusion of proteins across a liquid-liquid interface

Abstract A study of the rate of diffusion of some proteins across a liquid-liquid interfaces has been carried out using a two-phase system obtained by mixing aqueous solutions of the polymers polyethyleneglycol adn dextran. Six proteins with molecular weights in the range of 13 000–290 000 have been studied. Interfacial transfer coefficients and the corresponding values of free energy of activation have been determined. The results indicate that the rate of interfacial diffusion is a function of the partition coefficient as wella as the size of the diffusing molecule.

Hydrophobic surface properties of myosin in solution as studied by partition in aqueous two-phase systems: effects of ionic strength, pH and temperature

SummaryAffinity partitioning in dextran-polyethylene glycol-water biphasic systems has demonstrated that myosin has hydrophobic surface properties. In 0.5 M KCl at pH 7.5 myosin is transferred at increasing amounts to the polyethylene glycol-rich upper phase when an increasing proportion of that polymer in the system is replaced by its ester with lauric, myristic or palmitic acid. This shows that on its surface myosin has binding sites with affinity for long chain fatty acyl groups. Partition studies on the ionic strength range of 0.2–0.6 M KCl at pH 7.5 at 4°C and 20°C, respectively, in systems containing polyethylene glycol-palmitate showed that the affinity of myosin for the palmitate group becomes greater with (1) an increase in ionic strength, and (2) an increase in temperature at constant ionic strength. The affinity of myosin for the palmitate group also increases with a decrease in the pH in the range of 5.6 8.5. The increase in the affinity of myosin for the palmitate group parallels the increase in the tendency of myosin to self-interact and yield filaments.

Fractionation of chromosomes: I. A methodological study on the use of partition in aqueous two-phase systems and multiple sedimentation

Abstract Studies on the application of the techniques of counter-current distribution (CCD) in aqueous two-phase systems and multiple sedimentation for the fractionation of metaphase chromosomes are presented. The two-phase systems were composed of aqueous solutions of Dextran 500 and poly(ethylene)glycol 6000 (PEG). It has been found that different groups of chromosomes differ in their distribution between the two phases and that the introduction of PEG with covalently attached positively or negatively charged groups provides a means of steering the distribution of chromosomes. A rough fractionation of chromosomes on the basis of size is possible by the technique of multiple sedimentation and this, in combination with CCD, yields 10 fractions of chromosomes. Partition and CCD in aqueous two-phase system separate chromosomes according to their surface properties and may prove useful for isolation of individual chromosomes in bulk.

The 240-kDa subunit of human erythrocyte spectrin binds calmodulin at micromolar calcium concentrations.

Calmodulin (Human) Spectrin Erythrocyte cytoskeleton Aqueous two‐phase partitioning

Simplex optimization in biochemistry: Application of the method in two-phase partition

Partition in aqueous two-phase systems should have great potentialities for studies of biological material. However, the general use of the method has probably been hindered by the difficulties in finding the composition of two-phase systems with desired qualities. Experimental design has proved to be of immense value for optimization in many different areas and should also be useful in selecting the best possible two-phase system. Therefore the simplex method has been applied to the search for the composition of an aqueous two-phase system in which the partition behavior of filamentous actin and human spectrin differ as much as possible.

The rate of polymerization of rabbit skeletal muscle actin is enhanced by polyethylene glycol

SummaryThe effect of polyethylene glycol on the kinetics of actin polymerization was determined by monitoring the enhancement in the fluorescence of pyrenyl-labelled actin. The polymerization of actin at 15 mM KCl was in addition followed by viscometry and light scattering. All three methods showed that the overall rate of polymerization of actin increased 3-4-fold when the concentration of polyethylene glycol was increased from 0 to 6% (w w−1). A further increase in polyethylene glycol concentration to 10% (w w−1) caused a relatively small contribution to the increase in the rate of polymerization. The enhancement of the overall rate of polymerization by polyethylene glycol was also reflected in a significant decrease in the lag time observed when the time course of polymerization was followed by viscometry and light scattering. The steady-state value of fluorescence enhancement and critical concentration of actin were also influenced by polyethylene glycol and the results showed that the extent of polymerization was increased by an increase in the concentration of polyethylene glycol in solution. The effect of polyethylene glycol on both rate and extent of polymerization persisted at physiological salt concentration (150mm KCl, 2mm MgCl2). Since the rate of elongation was affected only to a small extent by polyethylene glycol, we propose that its main effect is on nucleation.