TAXOL (Fig. 1) was isolated from the plant Taxus brevifolia (western yew) by Wani et al., who reported that the molecule has antitumour activity in several experimental systems1. In our laboratory we have found that taxol, a low molecular weight neutral compound, completely inhibits division of exponentially growing HeLa cells at low concentrations of drug (0.25 µM) that have no significant effects on DNA, RNA or protein synthesis during a 4-h incubation with the cells. HeLa cells incubated with taxol for 20 h are blocked in late G2 and/or M (ref. 2). We report here that taxol acts as a promoter of calf brain microtubule assembly in vitro, in contrast to plant products such as colchicine and podophyllotoxin, which inhibit assembly. Taxol decreases the lag time for microtubule assembly and shifts the equilibrium for assembly in favour of the microtubule, thereby decreasing the critical concentration of tubulin required for assembly. Microtubules polymerised in the presence of taxol are resistant to depolymerisation by cold (4 °C) and CaCl2 (4 mM).

Promotion of microtubule assembly in vitro by taxol

The purpose of this review is to examine the various effects of low- molecular-weight electrolytes on the associations and interactions of proteins and nucleic acids. Our primary interest is in general electrostatic effects, rather than chemical effects (specific interactions) of particular ions (e.g. transition metals, protons). We consider those interactions in which a variation in salt concentration has a significant effect on the macromolecular equilibrium, and analyse the effects of salt in these situations in terms of (i) direct participation of ions in the biopolymer reaction, (ii) Debye—Huckel screening by salt ions of the charge interactions on the biopolymers, and (iii) the reduction in water activity brought about at high salt concentrations.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S003358350000202X

Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity.

Starting from known properties of non-specific salt effects on the surface tension at an air–water interface, we propose the first general, detailed qualitative molecular mechanism for the origins of ion-specific (Hofmeister) effects on the surface potential difference at an air–water interface; this mechanism suggests a simple model for the behaviour of water at all interfaces (including water–solute interfaces), regardless of whether the non-aqueous component is neutral or charged, polar or non-polar Specifically, water near an isolated interface is conceptually divided into three layers, each layer being 1 water-molecule thick We propose that the solute determines the behaviour of the adjacent first interfacial water layer ( I 1 ); that the bulk solution determines the behaviour of the third interfacial water layer ( I 3 ), and that both I 1 and I 3 compete for hydrogen-bonding interactions with the intervening water layer ( I 2 ), which can be thought of as a transition layer The model requires that a polar kosmotrope (polar water-structure maker) interact with I 1 more strongly than would bulk water in its place; that a chaotrope (water-structure breaker) interact with I 1 somewhat less strongly than would bulk water in its place; and that a non-polar kosmotrope (non-polar water-structure maker) interact with I 1 much less strongly than would bulk water in its place We introduce two simple new postulates to describe the behaviour of I 1 water molecules in aqueous solution The first, the ‘relative competition’ postulate, states that an I 1 water molecule, in maximizing its free energy (—δG), will favour those of its highly directional polar (hydrogen-bonding) interactions with its immediate neighbours for which the maximum pairwise enthalpy of interaction (—δ H ) is greatest; that is, it will favour the strongest interactions We describe such behaviour as ‘compliant’, since an I 1 water molecule will continually adjust its position to maximize these strong interactions Its behaviour towards its remaining immediate neighbours, with whom it interacts relatively weakly (but still favourably), we describe as ‘recalcitrant’, since it will be unable to adjust its position to maximize simultaneously these interactions The second, the ‘charge transfer’ postulate, states that the strong polar kosmotrope–water interaction has at least a small amount of covalent character, resulting in significant transfer of charge from polar kosmotropes to water–especially of negative charge from Lewis bases (both neutral and anionic); and that the water-structuring effect of polar kosmotropes is caused not only by the tight binding (partial immobilization) of the immediately adjacent ( I 1 ) water molecules, but also by an attempt to distribute among several water molecules the charge transferred from the solute When extensive, cumulative charge transfer to solvent occurs, as with macromolecular polyphosphates, the solvation layer (the layer of solvent whose behaviour is determined by the solute) can become up to 5- or 6-water-molecules thick We then use the ‘relative competition’ postulate, which lends itself to simple diagramming, in conjunction with the ‘charge transfer’ postulate to provide a new, startlingly simple and direct qualitative explanation for the heat of dilution of neutral polar solutes and the temperature dependence of relative viscosity of neutral polar solutes in aqueous solution This explanation also requires the new and intriguing general conclusion that as the temperature of aqueous solutions is lowered towards o °C, solutes tend to acquire a non-uniform distribution in the solution, becoming increasingly likely to cluster 2 water molecules away from other solutes and surfaces (the driving force for this process being the conversion of transition layer water to bulk water) The implications of these conclusions for understanding the mechanism of action of general (gaseous) anaesthetics and other important interfacial phenomena are then addressed

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0033583500005369

The Hofmeister effect and the behaviour of water at interfaces.

Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties These properties pose difficult stability problems A summary of both chemical and physical decomposition pathways for proteins is given Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species

Stability of Protein Pharmaceuticals

The stabilization of proteins by sucrose.

The effects of solution variables on the in vitro reconstitution of calf brain tubulin, purified by the method of Weisenberg et al. (Weisenberg, R. C., Borisy, G. G., and Taylor, E. W. (1968), Biochemistry 7, 4466-4479; Weisenberg, R. C., and Timasheff, S. N. (1970), Biochemistry 9, 4110-4116), as modified by Lee et al. (Lee, J. C., Frigon, R. P., and Timasheff, S. N. (1973), J. Biol. Chem. 248, 7253-7262), were investigated at pH 7.0. Reconstitution of microtubules was successful in a variety of buffer systems, the free energy of the propagation step of microtubule formation being little dependent on the buffer. Microtubule formation is promoted by magnesium ions and guanosine triphosphate, but inhibited by calcium ions. The dependence of the apparent association constant for microtubule formation on ligand concentration was analyzed by the linked function theory of Wyman (Wyman, J. (1964), Adv. Protein Chem. 19, 224-286), leading to the conclusion that the formation of a tubulin-tubulin contact involves the binding of one additional magnesium ion per tubulin dimer. Microtubule formation is also accompanied by the apparent binding of one additional proton and the release of water molecules, as suggested by the thermodynamic parameters determined. The reaction is entropy driven with an apparent heat capacity change, deltaCp, of -1500 +/- 500 cal/deg-mol. The enhancement of tubulin reassembly by glycerol is most likely due to nonspecific protein-solvent general thermodynamic interactions.

In vitro reconstitution of calf brain microtubules: effects of solution variables.

The in vitro reconstitution of calf brain tubulin, purified by the method of Weisenberg et al. [(1968), Biochemistry 7, 4466-4479; (1970), Biochemistry 9, 4110-4116] as modified by Lee et al. [(1973), J. Biol. Chem. 248, 7253-7262], was successful in a medium consisting of 10(-2) M sodium phosphate, 10(-4) M GTP, and concentrations of magnesium ions ranging from 0.5 to 16 X 10(-3) M at 37 degrees. Filaments resembling native microtubules were formed. The filaments are in equilibrium with the associating species of tubulin and the equilibrium can be shifted to depolymerization by lowering the temperature to 20 degrees. Filament formation is inhibited by calcium ions which also cause disassembly of the formed filaments. The effects of calcium ion can be reversed by the addition of [ethylenebis-oxyethylenenitrilo)]tetraacetic acid. The formation of filaments is favored by the presence of 3.4 M glycerol; only twisted abnormal filaments are observed in the presence of 1 M sucrose. The high molecular weight components observed in the sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of many tubulin preparations were shown not to be essential for the formation of the filaments.

The reconstitution of microtubules from purified calf brain tubulin.

The chemical characterization of calf brain microtubule protein subunits

A study has been carried out of the interactions with proteins of solvent systems which are commonly used to stabilize proteins in solution or to crystallize proteins out of solution. The first class consisted of water-sucrose and water-glycerol, which are frequently added to enzyme solutions in order to maintain their activity; the second class contained sodium and ammonium sulfates and 2-methyl-2,4-pentanediol (MPD) as solvent components. Densimetry and differential refractometry experiments with the application of multicomponent thermodynamic theory, showed that, in all four systems, proteins are preferentially hydrated, i.e., addition of these cosolvents results in an unfavorable free energy change. Detailed analysis of the thermodynamics of protein transitions in the presence of sucrose showed that its stabilizing action can be accounted for to a great extent in terms of the surface energy of this solvent system.

James C. Lee

Papers

In vitro reconstitution of calf brain microtubules: effects of solution variables.

The reconstitution of microtubules from purified calf brain tubulin.

The chemical characterization of calf brain microtubule protein subunits

The interaction of tubulin and other proteins with structure-stabilizing solvents☆

The stabilization of calf-brain microtubule protein by sucrose☆