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A range of carboxymethylated poly(hydroxyethyl methacrylate) (CM-PHEMA) hydrogels with varying degrees of carboxymethylation was synthesized for a systematic study of the effects of ionized groups (‘charge’) on the uptake by hydrogel matrices of the proteins, lysozyme and human serum albumin (HSA). Using a radiolabel-tracer technique, X-ray photoelectron spectroscopy, and laser scanning confocal microscopy, we attempted to differentiate between protein molecules that were irreversibly adsorbed onto the hydrogel surface and those that penetrated into the hydrogel matrix. The effective pore size of the CM-PHEMA hydrogels was modelled and compared with the known molecular dimensions of the two proteins. The effects of the presence of varying amounts of ionized groups in the hydrogel matrix differed for the two proteins. For lysozyme, increased uptake was observed at higher carboxymethylation; this is interpreted as resulting from a combination of electrostatic attraction and increasing ease of penetration of the protein into the more porous hydrogel matrix. For HSA, on the other hand, the uptake was primarily by surface adsorption, with little diffusive penetration into the matrix.

Optimization of strategies for the covalent attachment of proteins onto polymer surfaces requires the development of analytical methods which can differentiate between proteins that are covalently attached and those that are non-covalently bound (physisorbed). We probed for the surface density of reactive amine, carbonyl, and hydrazide groups using solution phase derivatization reactions to mimic and explore protein immobilization reaction strategies. Labeling compounds investigated were fluorescein derivatives, which were quantified by adsorption spectroscopy, and fluorinated phenyl compounds which were quantified by XPS. Control experiments consisted of performing the same labeling reactions using surfaces without reactive groups, or immersing the polymer surface into the labeling solution after blocking the reactive group of the labeling compound by a covalent reaction in solution. We always found non-negligible contributions arising from physisorption of the derivatization labels. Multiple control surfaces and a novel 'crossover derivatization-XPS' method were studied with the aim of improving compensation for physisorption. Our documentation of surprisingly large physisorption components even for small molecule labels, together with the known propensity of proteins to adsorb onto polymers, suggests caution in quantitative analysis of surface groups by derivatization, and in interpreting covalent protein immobilizations onto polymeric surfaces.

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy. In addition, a high density of proteins can be immobilized onto the reconditioned surfaces, since reactive carboxylic acids are regenerated by the procedure.

Fluoropolymers modified by plasma modification were studied for their suitability as surfaces for the adhesion of cells. We compared films made by plasma modification of fluoroethylenepropylene (FEP) using nitrogen-containing gases (ammonia or dimethyl acetamide) with films deposited using oxygen-containing monomers (methanol, methyl methacrylate or sequential treatment with toluene then water). The surfaces were compared for the attachment and spreading of human vein endothelial cells and human dermal fibroblasts. The initial attachment and spreading of cultured fibroblasts and endothelial cells onto films deposited using nitrogen-containing gases were equivalent to that onto films deposited using oxygen-containing monomers, but there were some differences in the mechanism of attachment. With films deposited using oxygen-containing monomers, the initial attachment and spreading of endothelial cells failed when the medium contained 15% (v/v) serum from which both fibronectin (Fn) and vitronectin (Vn) had been removed. Similarly, initial attachment and spreading of endothelial cells onto films deposited using oxygen-containing monomers were reduced by 62-86% when the cells were seeded in medium containing Vn-depleted serum (which contained Fn). Endothelial cells attached and spread onto films made using oxygen-containing monomers, when seeded in medium containing Fn-depleted serum (which contained Vn). On films deposited using nitrogen-containing gases, the adhesion of endothelial cells was only slightly reduced in Vn-depleted medium (as compared to attachment in medium containing unmodified serum). Furthermore, surfaces which had incorporated nitrogen were more effective than were oxygen-containing films in adsorbing sufficient serum Fn as to promote endothelial cell attachment. Similar results were seen for the attachment and spreading of fibroblasts as for the endothelial cells. For fibroblasts, attachment and spreading onto oxygen-containing films and onto nitrogen-containing films were not simply dependent upon either the Vn content or the Fn content of the medium. Maximal attachment and spreading of fibroblasts were, however, dependent upon adsorption of both serum Vn and Fn.

The attachment and growth of human endothelial cells and fibroblasts was studied on polymer surfaces fabricated by the polymerization of volatile amine and amide compounds in a low pressure gas plasma, and by the treatment of various surfaces in ammonia plasmas, which served to increase the nitrogen content of the surface layers. Infrared spectra showed the presence of amide groups, including those cases where the volatile compound ('monomer') did not contain oxygen. The performance of the surfaces in cell attachment correlated with the surface hydrophilicity and the nitrogen content, although for the latter a fair degree of scatter indicated that a more complex relationship applies. All these surfaces supported the attachment and growth of human cells. Generally, amide plasma polymers were best but the individual monomer and the plasma parameters also played a role. From comparisons of the various surfaces, it is suggested that the amide group is the main promoter of cell attachment in nitrogen-containing plasma surfaces.

Fluorescence quenching techniques have been used extensively in recent years to examine reaction rates and the compartmentalization of components in lipid micelles and membranes. Steady-state fluorescence methods are frequently employed in such studies but the interpretation of the resulting Stern-Volmer plots is often hampered by uncertainties regarding the mode of association of the quencher with the lipid structure and the nature of the quenching mechanism. This paper presents a method for simulating steady-state Stern-Volmer plots in two phase systems, and shows how the forms of such plots are influenced by the type of association of the quencher with the membrane or micelle (partition and/or binding) and by the type of quenching mechanism (dynamic and/or static). Comparisons of simulated plots with experimental data must take into account the possible combinations of quencher association(s) and quenching mechanism(s). The methods presented are applicable to synthetic and natural membranes and provide a basis for comparing the quenching of fluorescent molecules in biological membranes of differing composition.

Relations describing sedimentation equilibrium in solutions containing two macromolecular solute components are derived for the following cases: (1) two nonassociating proteins at arbitrary concentration, (2) one dilute self-associating protein in the presence of a second inert protein at arbitrary concentration, and (3) two proteins at arbitrary concentration that can associate to form a single heterocomplex of arbitrary composition. As in earlier work (R. C. Chatelier and A. P. Minton (1987) Biopolymers, 26, 507–524), the relations are obtained by using scaled particle theory to calculate the thermodynamic activity of each species present at a given radial distance in the centrifuge. The results of numerical simulations of sedimentation equilibrium are presented as the dependence of apparent molecular weights, or apparent weight-average molecular weights, upon solution composition. Semiempirical methods are presented, by means of which the weight-average molecular weights of self- and heteroassociating proteins in highly nonideal solutions may be estimated from experimental data. It is found that the semiempirical methods yield reasonably accurate estimates of the true weight-average molecular weight over a broad range of experimental conditions, providing that the partial specific volumes of two components in a heteroassociating system do not differ by more than about 0.05 mL/g.

The approach of a solution of dilute, monodisperse, globular macromolecules to low-speed sedimentation equilibrium in an ultracentrifuge is simulated by numerical integration of the Lamm equation. Various combinations of overspeed time and angular velocity are used to assess the conditions needed to minimize the time it takes the solution to attain sedimentation equilibrium. The optimal overspeeding time and angular velocity are determined over a wide range of values of the molecular weight (relative molar mass) of the solute and the radial distance between the meniscus and base of the solution. The results may be expressed as simple functions which allow facile calculation of (a) the optimal overspeeding time and velocity, and (b) the time required to reach sedimentation equilibrium. The results are in reasonable agreement with previous analytical solutions which were based on several simplifying assumptions. The parameterized overspeeding procedure is shown to be robust over a wide range of conditions, and typically leads to a greater than 5-fold reduction in centrifugation time.

To extract ligand-cell binding parameters from flow cytometric fluorescence data, one needs a method of converting the measured cellular fluorescence to the actual number of bound molecules producing that fluorescence. Bead standards containing a known number of fluorophores do not necessarily provide the correct conversion factor. We therefore present a method for calibrating flow cytometric bead standards. The technique uses the isoparametric analysis (Chatelier et al: EMBO J 5:1181--1186, 1986) to construct a plot of fluorescence per cell versus the number of bound ligands per cell, thus allowing a direct comparison of the quantum yields of the bead-associated fluorophore with that of the cell-bound fluorophore. The potential of this analysis is demonstrated by contrasting the fluorescent brightness of fluorescein isothiocyanate associated with thymocyte nuclei and synthetic polymer beads with that of fluoresceinated epidermal growth factor bound to A431 cells. When the standards are improperly calibrated, then the number of ligand-binding sites is incorrectly reported, and the derived Scatchard plot may exhibit an apparent positive or negative cooperativity as well as a strong dependence on cell concentration.

Consideration is given to the indefinite self-association of biological molecules assuming that each step-wise addition of monomer is characterized by the same enthalpy change, and that the second virial coefficients adequately describe the thermodynamic nonideality. Statistical thermodynamic models are used to calculate translational and rotational entropy changes accompanying the addition of each monomer to the aggregate. Three polymerization schemes are explored in which the solute species are modeled as right circular cylinders of various length/radius ratios. The analysis is applied to published data on the self-association of bovine liver glutamate dehydrogenase.

Equilibrium statistical-thermodynamic models are presented for the surface adsorption of proteins modeled as regular convex hard particles. The adsorbed phase is treated as a two-dimensional fluid, and the chemical potential of adsorbed protein is obtained from scaled particle theory. Adsorption isotherms are calculated for nonassociating and self-associating adsorbing proteins. Area exclusion broadens adsorption isotherms relative to the Langmuir isotherm (negative cooperativity), whereas self-association steepens them (positive cooperativity). The calculated isotherm for adsorption of hard spheres using scaled particle theory for hard discs agrees well with that calculated from the hard disc virial expansion. As the cross section of the adsorbing protein in the plane of the surface becomes less discoidal, the apparent negative cooperativity manifested in the isotherm becomes more pronounced. The model is extended to the case of simultaneous adsorption of a tracer protein at low saturation and a competitor protein with a different size and/or shape at arbitrary fractional saturation. Area exclusion by competitor for tracer (and vice versa) is shown to substantially enhance the displacement of tracer by competitor and to qualitatively invalidate the standard interpretation of ligand competition experiments, according to which the fractional displacement of tracer by competitor is equal to the fractional saturation by competitor.

Relations describing sedimentation equilibrium in solutions of self-associating macromolecules at arbitrary concentration are presented. These relations are obtained by using scaled-particle theory to calculate the thermodynamic activity of each species present at a given radial distance. The results are expected to be valid for solutions of globular proteins under conditions such that interactions between individual solute molecules may be approximated by a hard-particle potential. Sedimentation equilibria in solutions containing either a nonassociating solute or a solute that self-associates according to several different schemes are simulated using the derived relations. The results of these simulations are presented in terms of the dependence of apparent weight-average molecular weight upon solute concentration. Simple empirical relations are presented for estimating the true weight-average molecular weight from the apparent weight-average molecular weight, without reference to any particular self-association scheme. The weight-average molecular weight estimated in this fashion is within a few percent of the true weight-average molecular weight at all experimentally realizable solute concentrations ( < 400 g/L).