Allan H. Clark

Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH β-lactoglobulin gels

Heat-set gels and aggregates from beta-lactoglobulin (beta-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H(2)O as compared with D(2)O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse beta-sheet halo at 2θ=19 degrees in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm(-1) in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular beta-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, beta-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.

Enzyme specificity in galactomannan biosynthesis

Membrane-bound enzymes from developing legume-seed endosperms catalyse galactomannan biosynthesis in vitro from GDP-mannose and UDP-galactose. A mannosyltransferase [mannan synthase] catalyses the extension of the linear (1→4)-β-linked d-mannan backbone towards the non-reducing end. A specific α-galactosyltransferase brings about the galactosyl-substitution of the backbone by catalysing the transfer of a (1→6)-α-d-galactosyl residue to an acceptor mannosyl residue at or close to the non-reducing terminus of the growing backbone. Labelled galactomannans with a range of mannose/galactose (Man/Gal) ratios were formed in vitro from GDP-[14C]mannose and UDP-[14C]galactose using membrane-bound enzyme preparations from fenugreek (Trigonella foenum-graecum L.), guar (Cyamopsis tetragonoloba (L.) Taub.) and senna (Senna occidentalis (L.) Link.), species which in vivo, form galactomannans with Man/Gal ratios of 1.1, 1.6 and 3.3 respectively. The labelled galactomannans were fragmented using a structure-sensitive endo-(1→4)-β-d-mannanase and the quantitative fragmentation data were processed using a computer algorithm which simulated the above model for galactomannan biosynthesis on the basis of a second-order Markov chain process, and also the subsequent action of the endo-mannanase. For each galactomannan data-set processed, the algorithm generated a set of four conditional probabilities required by the Markov model. The need for a second-order Markov chain description indicated that the galactomannan subsite recognition sequence of the galactosyltransferase must encompass at least three backbone mannose residues, i.e. the site of substitution and the two preceding ones towards the reducing end of the growing galactomannan chain. Data-sets from the three plant species generated three distinctly different sets of probabilities, and hence galactose-substitution rules. For each species, the maximum degree of galactose-substitution consistent with these rules was closely similar to that observed for the primary product of galactomannan biosynthesis in vivo. The data provide insight into the mechanism of action and the spatial organisation of membrane-bound polysaccharide synthases.

Biopolymer Network Assembly: Measurement and Theory

Publisher Summary nPolymer networks are molecular-based systems whose network structure depends upon covalent or non-covalent interactions between macromolecules. The interactions can be simple covalent cross-links or more complex junction zone or particulate-type interactions. A number of biopolymer systems can self-assemble to form networks and gels and the assembly can occur by a variety of mechanisms. This chapter examines the nature of biopolymer gels and networks, the kinetics of assembly, and their characterization by rheological methods. The necessary theory to explain, for example, the complexities of gelation kinetics is then described. This chapter also discusses the nature of network assembly and the character of gels and their gelation. A number of traditional techniques have been used for gel measurements. They often have a major advantage in their low cost, compared to commercial apparatus. Nowadays the vast majority of physical measurements on gels are made using oscillatory shear rheometry. There must be easier ways of studying self-assembly in biopolymer systems than mechanical measurements, such as scattering and spectroscopic approaches. It is only by assembling and testing cure models against appropriate data that a link can be made between self-assembly and its mechanical implications. This is important as hydrated biopolymer networks form structural elements of living tissue.Publisher Summary Polymer networks are molecular-based systems whose network structure depends upon covalent or non-covalent interactions between macromolecules. The interactions can be simple covalent cross-links or more complex junction zone or particulate-type interactions. A number of biopolymer systems can self-assemble to form networks and gels and the assembly can occur by a variety of mechanisms. This chapter examines the nature of biopolymer gels and networks, the kinetics of assembly, and their characterization by rheological methods. The necessary theory to explain, for example, the complexities of gelation kinetics is then described. This chapter also discusses the nature of network assembly and the character of gels and their gelation. A number of traditional techniques have been used for gel measurements. They often have a major advantage in their low cost, compared to commercial apparatus. Nowadays the vast majority of physical measurements on gels are made using oscillatory shear rheometry. There must be easier ways of studying self-assembly in biopolymer systems than mechanical measurements, such as scattering and spectroscopic approaches. It is only by assembling and testing cure models against appropriate data that a link can be made between self-assembly and its mechanical implications. This is important as hydrated biopolymer networks form structural elements of living tissue.

CHAPTER 1 – Biopolymer Network Assembly: Measurement and Theory

Publisher Summary nPolymer networks are molecular-based systems whose network structure depends upon covalent or non-covalent interactions between macromolecules. The interactions can be simple covalent cross-links or more complex junction zone or particulate-type interactions. A number of biopolymer systems can self-assemble to form networks and gels and the assembly can occur by a variety of mechanisms. This chapter examines the nature of biopolymer gels and networks, the kinetics of assembly, and their characterization by rheological methods. The necessary theory to explain, for example, the complexities of gelation kinetics is then described. This chapter also discusses the nature of network assembly and the character of gels and their gelation. A number of traditional techniques have been used for gel measurements. They often have a major advantage in their low cost, compared to commercial apparatus. Nowadays the vast majority of physical measurements on gels are made using oscillatory shear rheometry. There must be easier ways of studying self-assembly in biopolymer systems than mechanical measurements, such as scattering and spectroscopic approaches. It is only by assembling and testing cure models against appropriate data that a link can be made between self-assembly and its mechanical implications. This is important as hydrated biopolymer networks form structural elements of living tissue.Publisher Summary Polymer networks are molecular-based systems whose network structure depends upon covalent or non-covalent interactions between macromolecules. The interactions can be simple covalent cross-links or more complex junction zone or particulate-type interactions. A number of biopolymer systems can self-assemble to form networks and gels and the assembly can occur by a variety of mechanisms. This chapter examines the nature of biopolymer gels and networks, the kinetics of assembly, and their characterization by rheological methods. The necessary theory to explain, for example, the complexities of gelation kinetics is then described. This chapter also discusses the nature of network assembly and the character of gels and their gelation. A number of traditional techniques have been used for gel measurements. They often have a major advantage in their low cost, compared to commercial apparatus. Nowadays the vast majority of physical measurements on gels are made using oscillatory shear rheometry. There must be easier ways of studying self-assembly in biopolymer systems than mechanical measurements, such as scattering and spectroscopic approaches. It is only by assembling and testing cure models against appropriate data that a link can be made between self-assembly and its mechanical implications. This is important as hydrated biopolymer networks form structural elements of living tissue.