Paul W. Chun

Use of water-soluble polymers for the isolation and purification of human immunoglobulins

Abstract By the use of fractional precipitation with high molecular weight nonionic polymers, immune globulins of marked homogeneity were isolated in high yield from either whole serum or commercial samples of immunoglobulins. The isolated fractions were characterized by immunochemical and ultracentrifugal analyses. When the pH was varied from 4.9 to 8.6 and the ionic strength from 0.1 to 2.0, little effect on the precipitation of the immunoglobulins was noted. The immunochemical studies indicated that this fractionation technique is not effective in separating the γ G , γ M , and γ A activities present in the γ-globulins isolated from whole serum or in commercial immunoglobulin preparations. Preliminary experiments indicate that this technique may be used for the isolation of other serum proteins, especially albumin and the α-globulins. In these instances, however, it is important that ionic strength and pH be controlled. The mechanism for the precipitation of serum proteins by PEG has not been established, but it is suggested that the precipitation involves a local dehydration and consequent change in the dielectric constant of the medium immediately surrounding the protein molecules. It would appear that the high-polymer precipitation technique may be an effective and simple method for the isolation of serum proteins with preservation of much of their native properties.

Effect of polyamines on the electrokinetic properties of red blood cells

Abstract At the physiological pH 7.4, the zeta potential of the normal red blood cell in 1.5% glycine buffer was found to be −52 mv, whereas that of sickling erythrocytes is −45 mv. Addition of spermidine to normal red blood cells reduced the zeta potential by approximately 20 mv. In sickling red blood cells, where the polyamine content is determined to be 5 to 6 times greater than in the normal erythrocyte, addition of spermidine reduced the zeta potential by only 5 mv, indicating that little more polyamine binding occurs. The polyamine content of whole blood taken from 24 patients having sickle cell anemia was found to be more than ten times that of whole blood from normal donors. Binding of polyamines to the normal red blood cell was analyzed from the surface charge potential variation as a function of polyamine concentration and the apparent binding constant determined to be 130 d1/g. The difference in the electrokinetic properties of normal and sickling red blood cells in this system may be attributed, in part, to a variation in the polyamine content of the two types of erythrocytes.

[22] Water-soluble nonionic polymers in protein purification

Publisher Summary Fractionation techniques employing nonionic water-soluble high polymers are of increasing significance in studying biologically important macromolecules and cell particulates. A two-phase system of dextran and polyethylene glycol (PEG) is employed in a countercurrent distribution procedure to separate two different strains of Escherichia coli. Several types of viruses, namely—bacteriophage, tobacco mosaic virus, vaccinia virus, and various polio and echo virus strains—are separated by distribution between two phases composed of buffers plus either dextran and PEG or dextran and methyl cellulose. PEG solutions are used to precipitate plant viruses and infectious bacteriophage particles are separated by sedimentation with PEG. An aqueous dextran and PEG two-phase system is employed to separate single-stranded DNA and double-stranded DNA, and the characteristics of the distribution of various RNA and DNA preparations in dextran and methyl cellulose systems are also measured.

A Thermodynamic Molecular Switch in Biological Systems: Ribonuclease S′ Fragment Complementation Reactions

Abstract It is well known that essentially all biological systems function over a very narrow temperature range. Most typical macromolecular interactions show ΔH° ( T ) positive (unfavorable) and a positive ΔS° ( T ) (favorable) at low temperature, because of a positive ( ΔCp°/T ) . Because ΔG° ( T ) for biological systems shows a complicated behavior, wherein ΔG° ( T ) changes from positive to negative, then reaches a negative value of maximum magnitude (favorable), and finally becomes positive as temperature increases, it is clear that a deeper-lying thermodynamic explanation is required. This communication demonstrates that the critical factor is a temperature-dependent ΔCp° ( T ) (heat capacity change) of reaction that is positive at low temperature but switches to a negative value at a temperature well below the ambient range. Thus the thermodynamic molecular switch determines the behavior patterns of the Gibbs free energy change and hence a change in the equilibrium constant, K eq , and/or spontaneity. The subsequent, mathematically predictable changes in ΔH° ( T ) , ΔS° ( T ) , ΔW° ( T ) , and ΔG° ( T ) give rise to the classically observed behavior patterns in biological reactivity, as may be seen in ribonuclease S′ fragment complementation reactions.[[page end]]

Exclusion of protein from high polymer media. I. Derivation of probability distribution for the number of fiber centers within any sphere of radius r.

Ogstons (1958) fiber model based on Poissons distribution function gives the average number of fibers making contact and no contact inside a sphere of radius r. The probability of penetration of spherical particles within a fibrous network was derived from the moment generating function [Formula: see text] A is the number of particles that intrude into a sphere of radious r. alpha(mu) is the probability that a particle, whose center is mu units away from the origin, intrudes into a sphere of radius r. A has a Poisson distribution with a mean value E(A) = 4pinualpha(mu)mu(2)dmu. The theoretical derivation of the distribution function of A gives Ogstons fiber model.

Thermodynamic molecular switch in micelles

Abstract It is well known that essentially all macromolecular interactions function over a well-defined temperature range. The Gibbs free energy change, ΔG°(T), for macromolecular interaction shows a complicated behavior, wherein ΔG°(T) changes from positive to negative, then reaches a negative value of maximum magnitude (favorable), and finally becomes positive as temperature increases. This communication demonstrates that the critical factor is a temperature-dependent ΔCp°(T) (specific heat capacity change) of reaction, which is positive at low temperature but switches to a negative value at a temperature well below the ambient range. This thermodynamic molecular switch determines the behavior patterns of the Gibbs free energy change, and hence a change in the equilibrium constant, Keq, and/or spontaneity. The subsequent, mathematically predictable changes in ΔH°(T), ΔS°(T), ΔW°(T) and ΔG°(T) give rise to the classically observed behavior patterns in biological systems. This communication will also demonstrate the existence of a thermodynamic molecular switch in both nonionic surfactants, (OPE)i where i=1, 3, 8, and 10 and ionic n-DTAB surfactants in H2O, D2O, 3 M urea and 2 M dioxane, over the experimental temperature range of 285–360 K, based on Chuns development of the Planck–Benzinger methodology (P.W. Chun, Int. J. Quantum Chem.: Quantum Biol. Symp. 15 (1988) 247; P.W. Chun, Manual for Computer-Aided Analysis of Biochemical Processes with Florida 1-2-4, University of Florida copyright reserved, 1991; P.W. Chun, J. Phys. Chem. 86 (1994) 6851; P.W. Chun, J. Biol. Chem., 270 (1995) 13925; P.W. Chun, J. Phys. Chem. 100 ( 1996) 7283; P.W. Chun, in: 212th National American Chemistry Society Meeting, Orlando, Fla, American Chemical Society, Biophysical Chemistry, poster 283, 1996; P.W. Chun, J. Phys. Chem. B 101 (1997) 7835; P.W. Chun, Methods in Enzymology, vol. 295, 1998, pp. 12, 227; P.W. Chun, Int. J. Quantum Chem.: Quantum Biol. Symp., 75 (1999) 1027; P.W. Chun, Int. J. Quantum Chem.: Quantum Biol. Symp. (2000); P.W. Chun, Biophysical J., 75 (2000) 416; P.W. Chun, Cell Biochem. Biophys. (2000)). In the case of micellar size distribution, the change in inherent chemical bond energy, ΔH°(T0), in micellar interaction is small. In contrast, the thermal agitation energy (heat capacity integrals), is much larger and roughly the same over a broad size distribution. This qualitative trend differs markedly from results seen for biochemical interactions, yet the underlying mathematical interpretation is the same.

Thermodynamic molecular switch in macromolecular interactions.

It is known that most living systems can live and operate optimally only at a sharply defined temperature, or over a limited temperature range, at best, which implies that many basic biochemical interactions exhibit a well-defined Gibbs free energy minimum as a function of temperature. The Gibbs free energy change, ΔGo (T), for biological systems shows a complicated behavior, in which ΔGo(T) changes from positive to negative, then reaches a negative value of maximum magnitude (favorable), and finally becomes positive as temperature increases The critical factor in this complicated thermodynamic behavior is a temperature-dependent heat capacity change (ΔCpo(T) of reaction, which is positive at low temperature, but switches to a negative value at a temperature well below the ambient range. Thus, the thermodynamic molecular switch determines the behavior patterns of the Gibbs free energy change, and hence a change in the equilibrium constant Keq, and/or spontaneity. The subsequent, mathematically predictable changes in ΔHo(T), ΔSo(T), ΔWo(T), and ΔGo(T) give rise to the classically observed behavior patterns in biological reactivity, as demonstrated in three interacting protein systems: the acid dimerization reaction of α-chymotrypsin at low pH, interaction of chromogranin A with the intraluminal loop peptide of the inositol 1,4,5-triphosphate receptor at pH 5.5, and the binding of l-arabinose and d-galactose to the l-arabinose binding protein of Escherichia coli. In cases of protein unfolding of four mutants of phage T4 lysozyme, no thermodynamic molecular switch is observed.

Interaction of human low density lipoprotein and apolipoprotein B with ternary lipid microemulsion. Physical and functional properties.

Based on data from sedimentation velocity experiments, electrophoresis, electron microscopy, cellular uptake studies, scanning molecular sieve chromatography using a quasi-three-dimensional data display and flow performance liquid chromatography (FPLC), models for the interaction of human serum low density lipoprotein (LDL) and of apolipoprotein B (apo B) with a ternary lipid microemulsion (ME) are proposed. The initial step in the interaction of LDL (Stokes radius 110 A) with the ternary microemulsion (Stokes radius 270 A) appears to be attachment of the LDL to emulsion particles. This attachment is followed by a very slow fusion into particles having a radius of approx. 280 A. Sonication of this mixture yields large aggregates. Electron micrographs of deoxycholate-solubilized apo B indicate an arrangement of apo B resembling strings of beads. During incubation, these particles also attach to the ternary microemulsion particles and, upon sonication, spherical particles result which resemble native LDL particles in size. Scanning chromatography corroborates the electron microscopy results. By appropriate choice of display angles in a quasi-three-dimensional display of the scanning data (corrected for gel apparent absorbance) taken at equal time intervals during passage of a sample through the column, changes in molecular radius of less than 10 A can be detected visually. Such a display gives a quantitative estimate of 101 +/- 2 A for these particles (compared to 110 A for native LDL). The LDL-ME particles and apo B-ME particles compete efficiently with native LDL for cellular binding and uptake. Cellular association studies indicate that both LDL- and apo B-ME particles are effective vehicles for lipid delivery into cells.

Scanning molecular sieve chromatography of interacting protein systems. II. Determination of large zone transport parameters by the difference profile method at low solute concentration.

The experimental determination of difference profiles for the study of large zone transport processes by scanning molecular sieve chromatography is described. Using the difference profile method, the progesterone-induced purple glycoprotein of the porcine uterus was found to exist as monomeric units in high ionic environment, with a partition coefficient of 0.269, partition cross-section of 0.488, partition radius of 25 A and a molecular weight of 33,500 g/mole. The technique was further applied in examining the association-dissociation properties of oxyhemoglobin. In a high tonic environment, the partition coefficient was found to be 0.365 for dimer and the partition cross-section, 0.419; for the tetramer in low ionic strength solution, the partition coefficient was 0.275 and the partition cross-section 0.377, with a dissociation constant of 1.03 x 10(-6) mole/l. This new technique should prove applicable in (1) readily locating the centroid positions of transport boundary profiles at the lowest practible protein concentration limits, (2) demonstrating the characteristic boundary shape and concentration-dependent centroid position for an interacting solute, (3) determining the axial dispersion coefficient characteristic of solute turbulence within the gel matrix, and (4) distinguishing the boundary between low and high ionic strength solvent phases in the gel column.

Molecular-Level Thermodynamic Switch Controls Chemical Equilibrium in Sequence-Specific Hydrophobic Interaction of 35 Dipeptide Pairs

Applying the Planck-Benzinger methodology, the sequence-specific hydrophobic interactions of 35 dipeptide pairs were examined over a temperature range of 273-333 K, based on data reported by Nemethy and Scheraga in 1962. The hydrophobic interaction in these sequence-specific dipeptide pairs is highly similar in its thermodynamic behavior to that of other biological systems. The results imply that the negative Gibbs free energy change minimum at a well-defined stable temperature, , where the bound unavailable energy, TdeltaS(o) = 0, has its origin in the sequence-specific hydrophobic interactions, are highly dependent on details of molecular structure. Each case confirms the existence of a thermodynamic molecular switch wherein a change of sign in deltaCp(o)(T)(reaction) (change in specific heat capacity of reaction at constant pressure) leads to true negative minimum in the Gibbs free energy change of reaction, deltaG(o)(T)(reaction), and hence a maximum in the related equilibrium constant, K(eq). Indeed, all interacting biological systems examined to date by Chun using the Planck-Benzinger methodology have shown such a thermodynamic switch at the molecular level, suggesting its existence may be universal.