Philip D. Ross

Analysis of non-ideal behavior in concentrated hemoglobin solutions.

Reported measurements of the concentration dependence of osmotic pressure and the sedimentation equilibrium of concentrated hemoglobin solutions may be quantitatively accounted for by a simple model which treats the solution as a suspension of hard quasispherical particles without long-range interactions. The results obtained do not preclude the presence of specific short-range attractive interactions between hemoglobin molecules. Model particles which best account for the experimental data are found to have approximately the same size, shape and mass as a hemoglobin molecule. The contribution of non-ideality to the total thermodynamic activity of hemoglobin in salt solution is shown to increase rapidly with increasing protein concentration.

Dye-ligand affinity chromatography: the interaction of Cibacron Blue F3GA with proteins and enzymes.

The dye Cibacron Blue F3GA has a high affinity for many proteins and enzymes. It has therefore been attached to various solid supports such as Sephadex, Sepharose, polyacrylamide, and the like. In the immobilized form the dye has rapidly been exploited as an affinity chromatographic medium to separate and purify a variety of proteins including dehydrogenases, kinases, serum albumin, interferons, several plasma proteins, and a host of other proteins. Such a diversity shown by the blue dye in binding several unrelated classes of proteins has generated considerable work in terms of studies of the chromophore itself and also the immobilized ligand. As a prelude to realizing the full potential of the immobilized Cibacron Blue F3GA, an understanding of the basic interactions of the dye with its surroundings must be gained. It has been recognized that the dye is capable of hydrophobic and/or electrostatic interactions at the instance of the ambient conditions. The study of interactions of the dye with salts, solvents, and other small molecules indicates the nature of the interactions of the dye with different kinds of groups at the interacting sites of proteins. The review will cover such interactions of the dye with the proteins, the interactions of the proteins with the immobilized ligand, and the media used to elute the bound protein in several cases, and thus consolidate the available information on such studies into a cogent and comprehensive explanation.

Hard quasispherical model for the viscosity of hemoglobin solutions

Abstract The viscosity of concentrated hemoglobin solutions of moderate ionic strength at pH values near the isoelectric point may be quantitatively described by the generalized form of a relation commonly applied to suspensions of hard spherical particles. This finding is consistent with the hard quasispherical model previously proposed to account for the thermodynamic properties of concentrated hemoglobin solutions under comparable conditions (1).

Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization

In HK97 capsid maturation, structural change (‘expansion’) is accompanied by formation of covalent crosslinks, connecting residue K169 in the ‘E‐loop’ of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead‐II passes through three expansion intermediate (EI) states en route to the end state, Head‐II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild‐type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (ΔTp, +11°C). EI‐II is the earliest capsid to form crosslinks. Cryo‐electron microscopy shows that for both wild‐type and K169Y EI‐II, most E‐loops are in the ‘up’ position, 30 Å from the nearest N356: thus, crosslinking in EI‐II represents capture of mobile E‐loops in ‘down’ positions. At pH 4, most K169Y capsids remain as EI‐II, whereas wild‐type capsids proceed to EI‐III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.

Inhibition of sickle cell hemoglobin gelation by some aromatic compounds

Summary Some simple organic molecules have been studied for their effects on the solubility of deoxyhemoglobin S. Compounds having an aromatic group with a pendant aliphatic polar moiety are the most effective in increasing the solubility. The results are rationalized in terms of a simple mechanism of non-covalent inhibition of gelation.

A calorimetric study of monomer-polymer complexes formed by polyribouridylic acid and some adenine derivatives.

Abstract This paper describes a calorimetric study of the reaction between various adenine derivatives with the common substrate polyribouridylic acid to form monomer-polymer complexes of the stoichiometry A:2 poly U † . A heat of reaction of −12.8 kcal./mole of A:2 poly U complex was found for the interaction between poly U and either adenine, adenosine or deoxyadenosine in 0.6 m -NaCl at 20 °C. This result indicates that the presence or absence of the sugar group or the 2′OH group contributes little to the ΔH of these monomer-polymer complexes. Complexes of poly U with 2-aminoadenosine and 2,6-diaminopurine, which can form three hydrogen bonds with the first strand of poly U, were found to be 3 kcal./mole more exothermic; that is, ΔH is −15.8 kcal./mole of A:2 poly U complex. These results were independently confirmed by direct calorimetric measurement of the energy absorbed in the melting of these complexes. It was found that the 2-amino derivatives are 3-kcal./mole more stable with respect to ΔH than the adenosine derivatives at their respective melting temperatures, T m . The standard entropy changes at T m calculated for dissociating these complexes are found to be large, positive and different for each system studied, with ΔS ° varying between 42 and 49 cal./deg. mole. It is suggested that the additional favorable enthalpy change accompanying the addition of the second polymer strand to form the 1:2 complex is decisive for overcoming the large unfavorable entropy change accompanying the immobilization of the monomer species upon incorporation into the 1:1 complex. This would account for the observation that monomer-polymer complexes are usually of 1:2 stoichiometry.

Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg(2+), DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 degrees C and 96 degrees C. In other conditions (absence of Mg(2+) and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit-probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.

Assembly-dependent conformational changes in a viral capsid protein: Calorimetric comparison of successive conformational states of the gp23 surface lattice of bacteriophage T4

Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (t*m) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23* prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta t*m = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23* in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.

The effect of non-aggregating proteins upon the gelation of sickle cell hemoglobin: Model calculations and data analysis

Abstract The scaled particle theory for mixtures of hard spheres is used to calculate the effect of added proteins of varying size upon the solubility of sickle cell hemoglobin. For a given added weight, smaller macromolecules are more effective in lowering the solubility of sickle cell hemoglobin. Calculations based upon this model agree with many recently reported observations. The observed effect of the addition of myoglobin or hemoglobin α-chains on the minimum gelling concentration of sickle cell hemoglobin (Benesch et al. ), however, is smaller than predicted. We suggest that this difference may arise from self-association of the added species.

A scanning microcalorimeter for thermally induced transitions in solution

Abstract A scanning microcalorimeter for the measurement of energies of transition in solution is described. The calorimeter utilizes semi-conductor thermoelectric modules and is of a very simple and inexpensive construction. The imprecision of measurement is three percent when measuring 25 to 250 mJ of heat associated with transitions over temperature intervals of up to 8 K. The calorimeter operates from ambient to 90°C.