Alan G. Walton

Structural changes in proteins adsorbed on polymer surfaces

The adsorption and desorption of plasma proteins—albumin, γ-globulin, and fibrinogen—on copolypeptide and silicone surfaces have been studied. The process is characterized by three stages; an initial brief period where adsorption is reversible; a second phase where the adsorbed proteins undergo a slow conformation change based on the period of adsorption and where protein is essentially irreversibly adsorbed; in the final stage, denatured material is slowly desorbed. In the second stage the process is essentially irreversible, desorption being very slow; the third stage is essentially irreversible, adsorption of denatured material being improbable. The adsorption process may be represented by a two-state Oreskes/Singer plot, there being an apparent surface phase change at 50–60% surface coverage. Albumin and fibrinogen molecules are adsorbed side-on; γ-globulin is adsorbed in an end-on configuration. Surface denaturation seems to be driven by a need to increase hydrophobic bonding with the surface and adsorption is heavily entropic in nature. In the desorbed material, albumin and fibrinogen sustain a marked decrease in α-helical content; γ-globulin loses most of its β-sheet structure.

Nucleation of Polymer Droplets

The nucleation of droplets of polyethylene, polyethylene oxide, polyoxymethylene, nylon 6, poly (3,3‐bis‐chloromethyloxacyclobutane), isotactic polypropylene, and isotactic polystyrene, suspended in silicone oil, has been investigated. Under conditions of slow cooling (0.1°C/min), two different phenomena were observed; occasional droplet solidification at low degrees of subcooling, the number of droplets solidified and the temperature at which nucleation occurred being a function of the thermal melt history; and a sudden catastrophic, history‐independent, nucleation phenomenon at large subcooling. The latter event may be identified as a critical phenomenon occurring at a subcooling ΔTc which is only slightly dependent upon the rate of cooling.For polyethylene and isotactic polypropylene, the ΔTcs correspond closely with values obtained by other authors using different suspending media and have been identified with homogeneous nucleation. Homogeneous nucleation may also be explored by the method of kineti...

A circular dichroism technique for the study of adsorbed protein structure

Abstract A cell that allows the structure of adsorbed protein layers to be examined by transmission circular dichroism is described. Sensitivity of the technique is demonstrated by presentation of spectra obtained from the adsorption of two blood proteins [bovine fibrinogen and human clotting factor XII (Hageman factor)] on a quartz substrate. The former does not undergo a conformational change on adsorption: the latter becomes activated and suffers a major structural change. It is suggested that such methodology is useful in the investigation of the interaction of blood components with prospective polymeric prosthetic implants.

The Secondary Structure of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents

Hageman factor (factor XII) is activated by exposure to surfaces such as glass or by solutions of certain compounds, notably ellagic acid. Changes in the structure of Hageman factor accompanying activation have been examined in this study by circular dichroism spectroscopy. The spectrum of unactivated Hageman factor in aqueous solutions suggests that its conformation is mainly aperiodic. Various perturbants altered the conformation of Hageman factor in differing ways, demonstrating the sensitivity of Hageman factor to its environment. After activation of Hageman factor with solutions of ellagic acid, a negative trough appeared in the region of the circular dichroism spectrum commonly assigned to tyrosine residues, along with other minor changes in the peptide spectral region. Some of these changes are similar to changes that occurred upon partial neutralization of the basic residues at alkali pH. Activation of Hageman factor by adsorption to quartz surfaces (in an aqueous environment) also produced changes similar to those in the ellagic acid-activated Hageman factor, including the negative ellipticity in the tyrosine region. These observations suggest that the activation process may be related to a change in status of some of the basic amino acid residues, coupled with a specific change in the environment of some tyrosine residues. The importance of these changes during the activation process remains to be determined. The sensitivity of Hageman factor to its environment is consistent with the view that the initiation of clotting by exposure of plasma to appropriate agents is brought about by alterations in the conformation of Hageman factor that occur in the apparent absence of Fletcher factor or other recognized clotting factors.

Application of fluorescence spectroscopy to the study of proteins at interfaces

Abstract A new technique for studying the nature of adsorbed protein molecules based on fluorescence spectroscopy is described. The method was used for following the adsorption of bovine serum albumin (BSA) onto particulates. On random copolypeptide substrates of the type (Glu(OBzl) x Ala y ), (Glu(OBzl) x Leu y ), or (Lys(CBZ) x Leu y ), considerable fluorescence quenching was observed. The origin of this effect appears to be a combination of the energy exchange between the excited tryptophan residue of BSA and the excited triplet state ( 3 Ba 2 b ) or of 1 L b singlet state of the substrate, and quenching by adjacent molecules. Since energy exchange of this type can occur over a distance of 100 A or so, it is possible to probe the protein environment of particles in the interfacial layer. It is concluded that even at low surface coverage, protein molecules are present adjacent to the surface in a nonadsorbed “methoric” layer.

Spectroscopic characterization of poly(Glu-Ala)

Abstract Infrared linear dichroism and ultraviolet circular dichroism speetroscopy have been used to distinguish four conformational forms of the ionizable sequential polypeptide poly(Glu-Ala). Two of these conformations, the α helix and the β form, were observed for the unionized polypeptide in solution. The α helix appeared immediately upon neutralization of the side-chain carboxyl functions, whereas the β form was observed after the neutralized solution had been standing for several days. The β form was also observed for films cast from either high or low pH solutions. Ionization of the glutamyl residues resulted in a circular dichroism spectrum which has previously been observed for charged homopolymers and appears to result from an extended helical conformation. Further, heating either the α helical or the charged extended helix resulted in a transition to a disordered chain. These results are consistent with the results of conformational calculations presented elsewhere.

Heterogeneous nucleation of water vapor on high and low energy surfaces

Abstract The rate of nucleation of water vapor condensing to drops on polycrystalline substrates having low and high surface energy was found to be strongly dependent upon the degree of supercooling. For each surface, a reproducible critical degree of supercooling was observed at which the nucleation rate changed rapidly. A plot of the critical degree of supercooling versus substrate surface energy gave a continuous curve with a minimum. The lowest degrees of supercooling were achieved by the silver halides. Materials having higher and lower surface energies than these halides gave greater critical degrees of super-cooling. For substrates of relatively low surface energy, the observed critical degree of super-cooling was generally smaller than the theoretical prediction of the Volmer-Becker-Doring heterogeneous nucleation theory indicating that the active nucleation sites must have a higher energy than the surface average. On high energy surfaces of cadmium and mercuric sulfide, where the contact angle was zero, the theory completely breaks down, and consequently predictions considerably underestimate the critical degree of supercooling. Variables not included in the theory such as the surface diffusion and adsorption free energies may account for these discrepancies.

Model polytripeptides for collagen

Abstract Poly[ala-glu(OEt)-gly], poly[glu(OEt)-gly-gly], and poly [ala-gly-gly] have been prepared from their respective tripeptide activated esters. Preliminary characterization by X-ray diffraction, infrared, circular dichroism, and optical rotatory dispersion spectroscopy indicate that poly[ala-glu(OEt)-gly], as obtained from an aqueous suspension or film cast from formic acid, is in an anti-parallel β sheet while the other two polymers are obtained in a random conformation from their aqueous solutions.

Epitaxial crystallization of poly (γ-benzyl L-glutamate) on alkali halide single crystals

Single crystals of poly(γ‐benzyl L‐glutamate) were formed by epitaxial crystallization from solution in mesitylene on NaCl, KI, and KCl (001) cleavage faces. From electron microscopy and diffraction studies, the structure of these overgrowths was determined to be that of lamellae containing chain‐folded α‐helical macromolecules. The usual type of crystal perfection, that of ordered helix axes and disordered side groups, was exhibited by this synthetic polypeptide. Unique orientation regimes were observed with each substrate.

Characterization of three sequential polydipeptides containing glycine

Abstract The three sequential polypeptides, poly(Ala-Gly), poly(Ser-Gly) and poly(Glu (OEt)-Gly) have been prepared and characterized using spectroscopic, X-ray diffraction and electron microscopic techniques. These three polymers may each be obtained in a fibrous crystalline form with a cross-β conformation. There is a systematic increase in the intersheet spacing as the methyl residue of alanine is replaced by more bulky residues. Infrared dichroism studies indicate that the molecular axis is perpendicular to the fiber axis and it is thought that antiparallel polymer chains arise from chain folding.