Jackie P. Healy

Novel approaches to the analysis of the Maillard reaction of proteins

The Maillard reaction comprises a complex network of reactions which has proven to be of great importance in both food science and medicine. The majority of methods developed for studying the Maillard reaction in food have focused on model systems containing amino acids and monosaccharides. In this study, a number of electrophoretic techniques, including two‐dimensional gel electrophoresis and capillary electrophoresis, are presented. These have been developed specifically for the analysis of the Maillard reaction of food proteins, and are giving important insights into this complex process.

Amyloid fibrils as functionalizable components of nanocomposite materials

Amyloid fibrils are a form of protein nanofiber that show promise as components of multifunctional bionanomaterials. In this work, native bovine insulin and bovine insulin that had been previously converted into amyloid fibrils were combined with poly(vinyl alcohol) (PVOH) via solution casting to determine the effect of fibrillization on the thermomechanical properties of the resulting composite. The synthesis method was found to preserve the amyloid fibril structure and properties of the resulting fibril‐PVOH composite were investigated. At a filling level of 0.6 wt %, the fibril‐reinforced PVOH was 15% stiffer than the PVOH control. Various properties of the films, including the glass transition temperature, degradation temperature, microstructure, and film morphology were characterized. Although more work is required to optimize the properties of the composites, this study provides proof of principle that incorporation of amyloid fibrils into a polymeric material can impart useful changes to the mechanical and morphological properties of the films.

Controlling the dimensions of amyloid fibrils: Toward homogenous components for bionanotechnology

Amyloid fibrils have been recognized as having potential in a variety of bionanotechnological applications. However, realization of these applications is constrained by a lack of control over morphology and alignment, both crucial for potential end uses. This article focuses on the use of growth and storage conditions to control the length of amyloid fibrils formed from bovine insulin, with length distributions constructed from transmission electron microscopy (TEM) images. Growth temperature, pH, protein concentration, and storage conditions were examined and were seen to offer a range of conditions that favor different length distribution. The use of amyloid fibrils as nanowires is one area where control of fibril dimensions is desirable, for experimental setup and endpoint applications. The conductive properties of fibrils formed from bovine insulin are presented, with these insulin fibrils being shown to have high resistivity in their unmodified state, with current values in the nanoamp range. These low current values can be increased via modification, or the fibrils used in their native state in applications where low current values are desirable. These findings, coupled with the ability to predict and select for various insulin amyloid fibril dimensions, enhances their utility as nanomaterials.

Polymorphism and higher order structures of protein nanofibers from crude mixtures of fish lens crystallins: Toward useful materials

Protein nanofibers are emerging as useful biological nanomaterials for a number of applications, but to realize these applications requires a cheap and readily available source of fibril-forming protein material. We have identified fish lens crystallins as a feedstock for the production of protein nanofibers and report optimized methods for their production. Altering the conditions of formation leads to individual protein nanofibers assembling into much larger structures. The ability to control the morphology and form higher order structures is a crucial step in bottom up assembly of bionanomaterials. Cell toxicity assays suggest no adverse impact of these structures on mammalian cell proliferation. There are many possible applications for protein nanofibers; here we illustrate their potential as templates for nanowire formation, with a simple gold plating process.

Aspects of physical and chemical alterations to proteins during food processing – some implications for nutrition

In this paper, we give an overview of our research exploring the impact of physical and chemical processing on food proteins. There are three themes, applied to the proteins of wheat, soya, egg and dairy foods. Firstly, the impact of the Maillard reaction on food proteins is discussed, with a particular focus on how the reactions might be harnessed to manipulate food texture. Secondly, the potential of enzymatic protein-protein crosslinking is considered, especially the enzyme transglutaminase. Thirdly, the broader question of how the aggregation of proteins within a food is altered by chemical and physical modification and how, in turn, this might impact on the overall nutritional quality of the food is considered.

Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white

The structural properties of amyloid fibrils combined with their highly functional surface chemistry make them an attractive new food ingredient, for example as highly effective gelling agents. However, the toxic role of amyloid fibrils in disease may cause some concern about their food safety because it has not been established unequivocally if consumption of food fibrils poses a health risk to consumers. Here we present a study of amyloid-like fibrils from whey, kidney bean, soy bean, and egg white to partially address this concern. Fibrils showed varied resistance to proteolytic digestion in vitro by either Proteinase K, pepsin or pancreatin. The toxicity of mature fibrils was measured in vitro and compared to native protein, early-stage-fibrillar protein, and sonicated fibrils in two immortalised human cancer cell lines, Caco-2 and Hec-1a. There was no reduction in the viability of either Caco-2 or Hec-1a cells after treatment with a fibril concentration of up to 0.25 mg/mL.

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid‐forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of “wavy” fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom‐up assembly of higher order protein structures from amyloid fibrils.

Development of a Practical Illustrating the Use of The Polymerase Chain Reaction for Genetic Testing

Introduction As a diagnostic tool for the detection of genetic and infectious diseases the polymerase chain reaction (PCR) has become very popular.~-3 The speed, reliability and potential for automation make PCR a powerful and robust technique that is already well established in laboratories where our biochemistry graduates find employment. The practical described here was adapted from the procedure developed for detecting a length polymorphism in the human proa2(I) collagen gene locus (COLIA2) by Watson and Dalgleish. 4 Its aims were to demonstratc PCR technique and its application to rapid genetic screening. The use of this particular diagnostic test includes the advantages that the students perform the experiment on their own genetic material with a resulting increase in the interest and relevance factors. In addition, tissue sampling is noninvasivc, painless and rapid, and the polymorphism detected is not associated with any known disease state 5 which removes most emotional and ethical problems associated with the experiment.