Does oxidation affect the water functionality of myofibrillar proteins?

Water-binding properties of myofibrils extracted from porcine muscle, and added hemoglobin with and without exposure to H2O2, were characterized using low-field proton NMR T2 relaxometry. The effects of pH and ionic strength in the samples were investigated as pH was adjusted to 5.4, 6.2, and 7.0 and ionic strength was adjusted to 0.29, 0.46, and 0.71 M, respectively. The formation of dityrosine as a measure of oxidative protein cross-linking revealed a significant increase in dityrosine concentrations upon H2O2 activation. The formation of dityrosine was strongly pH-dependent and increased with decreasing pH. In addition, increased levels of thiobarbituric acid reactive substances were observed upon addition of H2O2, implying that lipid oxidation was enhanced, however, with a different oxidation pattern as compared to the myofibrillar proteins. Low-field NMR relaxation measurements revealed reduced T2 relaxation times upon H2O2 activation, which corresponds to reduced water-holding capacity upon oxidation. However, a direct relationship between degree of oxidation and T2 relaxation time was not observed with various pH values and ionic strengths, and further studies are needed for a complete understanding of the effect of oxidation on myofibrillar functionality.     

Effects of supplemental‐nitrogen on the quality of rice proteins

A study was conducted on the effect of supplemental nitrogen (N) (20 hg/ha) applied as a foliar spray or to the soil on seed production, protein percentage, and protein fractions of rice. Plants were grown in a greenhouse over two different periods of time, i.e., August 1988 to January 1989 (Period I), and December 1988 to April 1989 (Period II). Nitrogen was applied to the leaves 10 and 20 days after anthesis (DAA), and to the soil at anthesis and at 15 DAA. Average temperature was 28.7°C during Period I and 32°C during Period II, corresponding to 18.7 and 22.0 growing degree‐day/day (GDD/day), respectively. The difference in GDD/day reduced the plant cycle from 130 days during Period I to 109 days during Period II. Plants grown during Period II had larger numbers of spikelets, a higher percentage of “full grown grains”;, and higher grain weight. Although percentage crude protein was about the same for the two periods, prolamin content was increased and the albumin+globulin fraction was decreased during Period II, but with no difference in glutelin content. The increase in number of spikelets, percent full grains, and grain weight appeared to result in a greater energy demand for plants grown during Period II. This may explain the increase in prolamins, since prolamin synthesis requires less energy than globulin or albumin synthesis. There was a simultaneous decrease in albumin and globulin synthesis during Period II. The content of glutelins, which represent the major reserve proteins in rice grains, was constant during both periods.     

Are molecular weights of proteins determined by superose 12 column chromatography correct?

Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.     

Interaction between β-casein and whey proteins as a function of pH and salt concentration

The effectiveness of β-casein as a chaperone in the aggregation of whey proteins was investigated. β-Casein altered heat-induced aggregation as shown by a reduction in turbidity of β-lactoglobulin, α-lactalbumin, and bovine serum albumin (BSA) solutions. The pH of the mixtures greatly affected how much β-casein reduced the turbidity of the solutions; the maximum reductions in turbidity were observed at pH 6.0. Reducing the pH decreased the effectiveness of β-casein as a chaperone. An increase in ionic strength by the addition of NaCl or CaCl(2) also decreased the effectiveness of the chaperone. The addition of CaCl(2) had a larger effect than the addition of NaCl. The chaperone effect was seen at temperatures up to 145 °C. Differential scanning calorimetry (DSC) showed that β-casein did not alter the denaturation temperature of β-lactoglobulin. The kinetics curves for loss of native protein and turbidity development showed that β-casein did not function by slowing the aggregation process. It was concluded that β-casein competes with whey protein in the aggregate process and the aggregates formed in the presence of β-casein are smaller in size than those formed during whey protein self-aggregation. The formation of smaller aggregates gives rise to less turbid, more soluble protein solutions.     

Proteolysis and bioconversion of cereal proteins to glutamate and γ-Aminobutyrate (GABA) in Rye malt sourdoughs

This study aimed to achieve the conversion of cereal proteins to the alternative end products glutamate or γ-aminobutyrate (GABA). Rye malt, fungal proteases, and lactobacilli were employed to convert wheat gluten or barley proteins. Glutamate and GABA formations were strain-dependent. Lactobacillus reuteri TMW1.106 and Lactobacillus rossiae 34J accumulated glutamate; L. reuteri LTH5448 and LTH5795 accumulated GABA. Glutamate and GABA accumulation by L. reuteri TMW1.106 and LTH5448 increased throughout fermentation time over 96 h, respectively. Peptides rather than amino acids were the main products of proteolysis in all doughs, and barley proteins were more resistant to degradation by rye malt proteases than wheat gluten. However, addition of fungal protease resulted in comparable degradation of both substrates. Glutamate and GABA accumulated to concentrations up to 63 and 90 mmol kg(-1) DM, respectively. Glutamate levels obtained through bioconversion of cereal proteins enable the use of hydrolyzed cereal protein as condiment.     

New analogues of (E)-β-farnesene with insecticidal activity and binding affinity to aphid odorant-binding proteins

(E)-β-farnesene is a strong and efficient alarm pheromone in most aphid species. However, applications in agriculture are prevented by its relatively high volatility, its susceptibility to oxidation and its complex and expensive synthesis. To develop novel compounds for aphid control, we have designed and synthesized analogues of (E)-β-farnesene, containing a pyrazole moiety present in several insecticides. Their structures have been confirmed by 1H NMR, elemental analysis, high-resolution mass spectroscopy and IR. Binding activities to three odorant-binding proteins (OBPs) of the pea aphid Acythosiphon pisum have been evaluated and correlated with their structures with reference to (E)-β-farnesene. Several derivatives were shown both to bind to A. pisum OBPs with a specificity similar to that of (E)-β-farnesene and to have aphicidal activity comparable to that of thiacloprid, a commercial insecticide. The compounds synthesized in this work represent new potential agents for aphid population control and provide guidelines to design analogues of (E)-β-farnesene endowed with both insecticidal and repellent activity for aphids.     

Reaction of the butter flavorant diacetyl (2,3-butanedione) with N-α-acetylarginine: a model for epitope formation with pulmonary proteins in the etiology of obliterative bronchiolitis

The butter flavorant diacetyl (2,3-butanedione) is implicated in causing obliterative bronchiolitis in microwave popcorn plant workers. Because diacetyl modifies arginine residues, an immunological basis for its toxicity is under investigation. Reaction products of diacetyl with N-α-acetylarginine (AcArg) were determined as a model for hapten formation, with characterization by mass spectrometry, NMR, and HPLC with UV detection and radiodetection. Four products were identified by LC-MS, each with a positive ion of m/z 303 (diacetyl + AcArg); one pair displayed an additional ion at m/z 217 (AcArg), the other pair at m/z 285 (- H(2)O). Their (1)H-(13)C NMR correlation spectra were consistent with the addition of one or two of the guanidine nitrogens to form aminols. Open-chain pairs interconverted at pH 2, as did the cyclized, but all four interconverted at neutral pH. This is the first structural characterization of the covalent adducts between diacetyl and an arginine moiety.