Preparation of self-assembled microspheres and their potential for drug delivery.

Dextran solutions intended for use as plasma extenders have been observed to form insoluble precipitates. Earlier studies of precipitation have shown that in solutions of 50% and 60% w/w of dextran molecular mass 6000 g mol(-1) beaded precipitates are formed over a two-week period. This study considers dextran precipitation over a wider molecular mass range and the kinetics, of formation, morphology and potential utility of these precipitates is investigated. Results show precipitation occurs over the dextran molecular mass range 6000-17,000 g mol(-1), with lower molecular mass material showing more rapid precipitation. As bead formation is accompanied by an increase in turbidity, formation kinetics were quantified spectrophotometrically confirming that precipitation rates were inversely proportional to molecular mass. The utility of these precipitates for drug delivery applications was assessed using bovine serum albumin as a protein drug analogue. The results showed that the inclusion of protein did not prevent bead formation and that entrapped protein was subsequently released from dextran beads in a time dependant manner. This suggests that dextran beads of this type may find application in the drug delivery area, as they combine the advantages of mild entrapment conditions with the use of an unmodified clinically approved polymer.

The effect of temperature on enzyme activity: new insights and their implications.

The two established thermal properties of enzymes are their activation energy and their thermal stability. Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive transition, in addition to an irreversible thermal inactivation. Two new thermal parameters, Teq and Delta Heq, describe the active-inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We review here the Model itself, methods for the determination of Teq and Delta Heq, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

Catalytic efficiency and kcat/KM: a useful comparator?

The ratio k(cat)/K(M)--often referred to as the "specificity constant"--is a useful index for comparing the relative rates of an enzyme acting on alternative, competing substrates. However, an alternative description, "catalytic efficiency", is frequently used, and on occasions misused, to compare the reactivity of two enzymes acting on the same substrate. Here, we highlight the pitfalls in using k(cat)/K(M) to compare the catalytic effectiveness of enzymes.

Eurythermalism and the temperature dependence of enzyme activity.

The "Equilibrium Model" has provided new tools for describing and investigating enzyme thermal adaptation. It has been shown that the effect of temperature on enzyme activity is not only governed by deltaG(double dagger)(cat) and deltaG(double dagger)(inact) but also by two new intrinsic parameters, deltaH(eq) and T(eq), which describe the enthalpy and midpoint, respectively, of a reversible equilibrium between active and inactive (but not denatured) forms of enzyme. Twenty-one enzymes from organisms with a wide range of growth temperatures were characterized using the Equilibrium Model. Statistical analysis indicates that T(eq) is a better predictor of growth temperature than enzyme stability (deltaG(double dagger)(inact)). As expected from the Equilibrium Model, deltaH(eq) correlates with catalytic temperature tolerance of enzymes and thus can be declared the first intrinsic and quantitative measure of enzyme eurythermalism. Other findings shed light on the evolution of psychrophilic and thermophilic enzymes. The findings suggest that the description of the Equilibrium Model of the effect of temperature on enzyme activity applies to all enzymes regardless of their temperature origins and that its associated parameters, deltaH(eq) and T(eq), are intrinsic and necessary parameters for characterizing the thermal properties of enzymes and their temperature adaptation and evolution.

A smart membrane based on an antigen-responsive hydrogel.

Hydrogel membranes have been fabricated that incorporate antibody/antigen moieties. The permeability of large solutes through these membranes is dependent on the presence of soluble antigen that can compete with the internal interactions between antibody and antigen leading to an increase in gel mesh size. Specifically, the membrane's structure is based on a dextran backbone grafted with a fluorescein isothiocyanate (FITC) antigen and a sheep anti-FITC IgG antibody. The backbone is covalently cross-linked by conjugated divinyl sulfone (DVS) groups. The gel structure is additionally stabilized by affinity crosslinks formed by biospecific interactions between the bound IgG and FITC. FTIR spectra of the gel are consistent with formation of covalent bonds between cysteine groups in the IgG and DVS groups in the dextran. Results obtained using isothermal titration calorimetry (ITC) confirmed the competitive interaction binding between IgG-FITC-dextran and free sodium fluorescein at pH 5.0. Scanning electron microscopy (SEM) of samples prepared using cryofixation and cryofracturing techniques showed that observed changes in permeability correlate with free fluorescein-dependent structural changes in the gel. Three-dimensional images obtained from confocal laser scanning microscopy show that these changes occur throughout the gel and indicate that SEM results are not artifacts of sample preparation. The permeability of these gels, as shown by blue-dextran (12 kDa) diffusion, increases in response to the presence of free fluorescein of the external medium, which causes competitive displacement of the affinity cross-links. Sequential addition and removal of sodium fluorescein showed that these permeability changes are reversible.

The dependence of enzyme activity on temperature: determination and validation of parameters.

Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by DeltaG(Dagger)(cat), and irreversible inactivation defined by DeltaG(Dagger)(inact). However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E(inact)) that is in reversible equilibrium with the active form (E(act)); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, DeltaH(eq), and a new thermal parameter, T(eq), which is the temperature at which the concentrations of E(act) and E(inact) are equal; T(eq) may therefore be regarded as the thermal equivalent of K(m). Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T(eq) and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.

The thermal behaviour of enzyme activity: implications for biotechnology.

The way that enzymes respond to temperature is fundamental to many areas of biotechnology. This has long been explained in terms of enzyme stability and catalytic activation energy, but recent observations of enzyme behaviour suggest that this picture is incomplete. We have developed and experimentally validated a new model to describe the effect of temperature on enzymes; this model incorporates additional fundamental parameters that enable a complete description of the effects of temperature on enzyme activity. In this article, we consider the biotechnological implications of this model in the areas of enzyme engineering, enzyme reactor operation and the selection and/or screening of useful enzymes from the environment.

An intrinsically shielded hydrogel for the adsorptive recovery of lysozyme.

The present paper addresses the selective recovery of lysozyme from egg white using CM-dextran (carboxymethyldextran)-based hydrogels containing Cibacron Blue as an affinity ligand and co-immobilized BSA intended to act as a shielding agent to reduce non-specific adsorption. Initial studies using pure lysozyme were conducted that indicated that the adsorption capacity increased with ligand density and that adsorption was well described by a Langmuir-type isotherm. The inclusion of BSA as a putative shielding agent did not decrease the adsorption capacity for lysozyme in single-adsorbate experiments. To assess the effectiveness of the shielding strategy, subsequent experiments were conducted with both defined lysozyme/ovalbumin mixtures and hen's-egg white. From these studies, the optimal operating conditions for lysozyme recovery have been determined. These include: optimal initial egg-white concentration [a 10% (v/v) solution of native egg white in the chosen buffer], affinity-ligand density (1.86 mM) and ligand-to-shielding-agent ratio (4:1). The purity of lysozyme obtained from egg white was improved from 69% with a non-shielded hydrogel to 94% with an intrinsically shielded hydrogel. Finally, the possibility of using a protein, rather than dextran-backbone-based, hydrogel was investigated. It was found that BSA could take the place of CM-dextran as the gel backbone in a simplified synthesis, producing a gel which also proved effective for lysozyme recovery with a 30% lysozyme in egg-white solution purified to approx. 92% in a single adsorption-desorption cycle.

Molecular characterization of human xanthine oxidoreductase: the enzyme is grossly deficient in molybdenum and substantially deficient in iron-sulphur centres.

XOR (xanthine oxidoreductase) purified from human milk was shown to contain 0.04 atom of Mo and 0.09 molecule of molybdopterin/subunit. On the basis of UV/visible and CD spectra, the human enzyme was approx. 30% deficient in iron-sulphur centres. Mo(V) EPR showed the presence of a weak rapid signal corresponding to the enzyme of low xanthine oxidase activity and a slow signal indicating a significant content of desulpho-form. Resulphuration experiments, together with calculations based on enzymic activity and Mo content, led to an estimate of 50-60% desulpho-form. Fe/S EPR showed, in addition to the well-known Fe/S I and Fe/S II species, the presence of a third Fe/S signal, named Fe/S III, which appears to replace partially Fe/S I. Comparison is made with similarly prepared bovine milk XOR, which has approx. 15-fold higher enzymic activity and Mo content. Taken along with evidence of low Mo content in the milk of other mammals, these findings add further support to the idea that XOR protein plays a physiological role in milk (e.g. in secretion) equal in importance to its catalytic function as an enzyme.

A novel pH- and ionic-strength-sensitive carboxy methyl dextran hydrogel.

A fast and simple method for the preparation of pH-sensitive hydrogel membranes for drug delivery and tissue engineering applications has been developed using carbodiimide chemistry. The hydrogels were formed by the intermolecular cross-linking of carboxymethyl dextran (CM-dextran) using 1-ethyl-(3-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Infrared spectra of the hydrogels suggest the formation of ester bonds between the hydroxyl and carboxyl groups in the CM-dextran. The porosity of the hydrogels produced, as shown by protein diffusion, increases in response to changes in the pH and the ionic strength of the external medium. The results show pH-dependent swelling behaviour arising from the acidic pedant groups in the polymer network. The diffusion of the protein lysozyme through the hydrogel membranes increased with increases in both pH (5.0-9.0) and ionic strength. The effect of changes of pH and ionic strength on the hydrogel's permeability was shown to be reversible. Scanning electron microscopy of these hydrogels showed that pH-dependent changes in permeability are mirrored by morphological changes in gel structure.

A new intrinsic thermal parameter for enzymes reveals true temperature optima.

Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, T(eq), arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source ("evolved") temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, T(eq) is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on T(eq) with respect to enzyme temperature/activity effects to be more important than on thermal stability. T(eq) is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution.

Bubble-induced detachment of affinity-adsorbed erythrocytes.

It is desirable that cells adsorbed in affinity-separation processes be easily recovered from the adsorption surface, without excessive dilution, once contaminants have been removed. The present study investigates the use of gas-bubble-induced shear stress for the recovery of affinity-adsorbed human erythrocytes. This method has previously been demonstrated to be effective with yeast cells, where it allows cells to be attached, washed and detached under isocratic conditions. Concanavalin A (Con A), used as the binding agent, was attached to the inside of nylon tubes. Whole blood solution, diluted to an erythrocyte concentration of 1x10(8) x ml(-1) with PBS, was incubated with the Con A-nylon surface and then washed with PBS prior to elution. To effect elution, air bubbles of known volume were introduced to the buffer feed to the tubes and the effects of bubble size, bubble volume and bubble velocity on detachment being determined. The results obtained showed that the most significant parameter was bubble number, with up to 90% of attached cells being recovered using a five-bubble sequence. Microscopic examination showed no evidence of mechanical damage to the detached cells.

Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

Antimicrobial properties of milk: dependence on presence of xanthine oxidase and nitrite.

Human and bovine milk inhibited the metabolic activity of Escherichia coli, as shown by luminescence monitoring of constructs expressing the luxCDABE genes. Inhibition was dependent on both xanthine oxidase (XO) activity and on the presence of nitrite, implying that XO-generated nitric oxide functions as an antibacterial agent.