Peter D. Hoagland

Secondary structural studies of bovine caseins: temperature dependence of β-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization

Abstract To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between β-casein and the other caseins, three-dimensional models have been presented. Secondary structural prediction algorithms and molecular modeling techniques were used to predict β-casein structure. The secondary structure of bovine β-casein was re-examined using Fourier transform infrared and circular dichroism spectroscopies to test these predictions. Both methods predict a range of secondary structures for β-casein (28–32% turns, 32–34% extended) at 25°C. These elements were highly stable from 5 to 70°C as viewed by circular dichroism. More flexible conformational elements, tentatively identified as loops, helix and short segments of polyproline II, were influenced by temperature, increasing with elevated temperatures. Another view is that as temperature decreases, these elements are lost (cold denaturation). Several distinct transitions were observed by circular dichroism at 10, 33 and 41°C, and another transition, extrapolated to occur at 78°C. Calculations from analytical ultracentrifugation indicate that the 10, 33 and 41°C transitions occur primarily in the monomeric form of the protein. As β-casein polymers are formed, and increase in size, the transitions at higher temperature may reflect changes in the more flexible conformational elements as they adjust to changes in surface charge during polymer formation. The transition at 10°C may represent an actual general conformational change or cold denaturation. Over the range of temperatures studied, the sheet and turn areas remain relatively constant, perhaps forming a supporting hydrophobic core for the monomers within the micelle-like polymer. This interpretation is in accord with the known properties of β-casein, and those predicted from molecular modeling.

Characterization of pectin, flash-extracted from orange albedo by microwave heating, under pressure.

Pectin was acid extracted from orange albedo by microwave heating under pressure. Extraction times ranged from 2.5 to 8 min. Solubilized pectin was characterized for molar mass (M), rms radius of gyration (Rg) and intrinsic viscosity [eta] by HPSEC with online light scattering and viscosity detection. M, Rg and [eta] all decreased with increasing extraction time. Nevertheless, at heating times of 2.5 and 3.0 min, M, Rg and [eta] were significantly higher than a commercial citrus pectin when the albedo:solvent ratio was 1:25 (w/v). At the heating time of 2.5 min Mw was 3.6 x 10(5), Rgz was 38 nm and [eta]w was 10.8 dL/g. Chromatography revealed that solubilized pectin distributions were bimodal in nature and that the low-molar-mass fraction increased at the expense of the high-molar-mass fraction with increasing extraction time. Scaling law exponents revealed that the high-molar-mass fraction was extremely compact in shape, whereas the low-molar-mass fraction was more asymmetric in shape. Possibly these results indicated that at short extraction times, pectin was solubilized as compact aggregated network structures that were broken down to their more asymmetric components with increased heating times.

Environmental Influences on Bovine κ-Casein: Reduction and Conversion to Fibrillar (Amyloid) Structures

The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of κ-casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the κ-casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem.17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-κ). Surprisingly, at only 37°C, the RCM-κ-casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM-κ-caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and κ turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in κ-casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM-κ-casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.

Characterization of starches dissolved in water by microwave heating in a high pressure vessel

Starch granules derived from four corn varieties were dispersed in water and depending on variety about 49–71% of the granules dissolved by microwave heating in a high pressure vessel (MWHPV). The apparent ratios of amylopectin to amylose were 1:0, 3:1, 1:1 and 3:7. High performance size exclusion chromatography (HPSEC) was carried out using two μ Bondagel† and one Synchropak† HPSEC columns placed in series. These had size exclusion limits specified by their manufacturers as 400 nm, 100 nm, and 10 nm, respectively. The mobile phase was 0.05 m NaNO3. For each starch composition, refractive index and viscosity chromatograms were obtained and fitted with the same six Gaussian components by nonlinear regression analysis. Calibration of the column set with pullulan and dextran standards in hydrodynamic volume and root mean square radius of gyration (Rg) enabled calculation of the intrinsic viscosity (IV), molecular weight (M), and Rg for each component in addition to global values of these quantities for the entire distribution. Analysis of the data revealed that as starches eluted from the column set, there were large changes in M and Rg and rather small changes in IV. Furthermore, MWHPV containing water as employed here produces starch of relatively large molecular weight and size but low intrinsic viscosity leading to the conclusion that dense starch granule fragments were solubilized.

The formation of intermediate lactones during aminolysis of diethyl galactarate

Abstract The aminolysis of diethyl galactarate has been found to proceed through intermediate γ-lactones. In dimethyl sulfoxide at 31°, the 1,6-diester is quickly converted into the 6-ester 1,4-lactone through base catalysis, and this lactone reacts with a primary amine to yield a 6-ethyl galactaric 1-amide that rapidly affords the 6,3-lactone, which reacts with the amine to give the galactaric diamide. The reaction sequence was established by 13 C-n.m.r. spectroscopy, which suggested competitive, consecutive, second-order kinetics in which k 1 = k 2 can be applied.

Screening the physical properties of novel Pseudomonas exopolysaccharides by HPSEC with multi-angle light scattering and viscosity detection

The physical properties of three novel acidic exopolysaccharides obtained from P. marginalis types A, B and C, one from P. ‘gingen’, one from P. andropogenis and one from P. fluorescens have been partially characterized. These EPSs were chromatographed on three serially placed SE Shodex OH pak columns covering a molar mass range for pullulans from about 4 × 107 to 1 × 103. The mobile phase was 0.05 M NaNO3. Physical measurements were performed on about 30 mg of sample for each EPS. The weight average molar mass of these EPSs ranged from about 0.71 to 2.85 × 106, the weight average intrinsic viscosity from 7.15 to 35.3 dl/ g and the radius of gyration from 62 to 123nm. The polydispersities of these EPSs ranged from 1.01 to 1.37. The large molar mass, size and viscosities of these EPSs may indicate that they have potential for use as thickeners, stabilizers, emulsifiers, and gelling agents in the food and non-food industries.

Characterization of Hemicellulose B from Corn Fiber by High-Performance Size Exclusion Chromatography with On-Line Molar Mass and Viscometric Detection

Abstract Hemicellulose B (arabinoxylan, corn fiber gum, CFG) was prepared from corn fiber (CF) by alkaline extraction with 2 meq/g of CF using either NaOH, Ca(OH)2 or an equimolar mixture of the two. Prior to CFG extraction, oil was removed from CF by extraction with hexane and starch was removed from CF by treatment with α-amylase. The solutions of alkali-extracted CFG were then bleached with alkaline H2O2. The effect of extractant composition on the weight-average molar mass M w, weight-average intrinsic viscosity [η]w, and z-average root-mean-square radius of gyration R gz on CFG was determined. Depending on extractant composition and method of calculation, M w ranged from 2.7–6.96 × 105, [η]w from 1.87 to 2.00 dL/g and R gz from about 31 to 50 nm. Substituting Ca(OH)2 for NaOH reduced M w and R gz but had little or no effect on [η]w. Chromatographic analysis in conjunction with molar mass detection revealed that molar mass distributions were bimodal. M w for the high-molar-mass component ranged from about 8.4–16.1 × 105, whereas M w ranged from about 1.1–2.1 × 105 for the low-molar-mass component. The high-molar-mass component comprised about 15–43% by weight of CFG and was much more compact in shape than the low-molar-mass component.

Reductive amination of lactose: unusual 13C-N.M.R. spectroscopic properties of N-alkyl-(1-deoxylactitol-1-yl)amines☆

Abstract Lactose was reductively aminated with selected alkylamines and sodium cyanoborohydride in boiling methanol in the presence of a weak organic acid. In alkaline solution, the N-alkyl-(1-deoxylactitol-1-yl)amines exhibited unusual behavior that was reflected by peak splitting in the 13C-n.m.r. spectra of the β- d -galactopyranosyl- and d -glucitol-1-yl C-1 resonances. Anisotropic effects, documented by longitudinal relaxation-time measurements, suggest that motion in the carbohydrate region of the N-alkyl-(1-deoxylactitol-1-yl)amines is restricted by intermolecular interactions at high pH.

The determination of free ϵ-amino groups of lysine in proteins using 19F NMR spectroscopy☆

Abstract The amount of free ϵ-amino groups of lysine residues in proteins can be determined by a fast and straightforward method based on the use of 19F NMR spectroscopy. Unsubstituted ϵ-amino groups are trifluoroacetylated with the reagent, S-ethyl trifluorothioacetate, in dimethyl sulfoxide solution, and the number of such groups is quantitatively determined using standard 19F NMR techniques. The method is evaluated using the following proteins: α-lactalbumin, β-lactoglobulin, bovine serum albumin, β-casein, and lysozyme.

Simulation of diffusion-limited aggregation of pectin

Abstract End-to-end diffusion-limited aggregation of an ideal, monodisperse pectin species with variable probability of interrmolecular crosslinking has been simulated on a lattice. The aggregate was fixed on the lattice as a structure of segmented rods. The growth of averages of size, length, width, and radius of gyration followed a power law dependence on time as measured by number of random lattice movements of diffusing monomer. The structures exhibited multifractal properties that arise from both the diffusion process and the random generation of crosslinking sites. The growth of the average length of the structures was independent of probability of crosslinking under the condition of constant presence of diffusing monomer. The growth of the average size of the structures was directly related to the growth of the average number of crosslinks. Average length, width, and radius of gyration were related to growth of average size by power laws. Macintosh and IBM PC source code and executables are included on disk in this issue.