John Markwell

Calibration of the Minolta SPAD-502 leaf chlorophyll meter

Use of leaf meters to provide an instantaneous assessment of leaf chlorophyll has become common, but calibration of meter output into direct units of leaf chlorophyll concentration has been difficult and an understanding of the relationship between these two parameters has remained elusive. We examined the correlation of soybean (Glycine max) and maize (Zea mays L.) leaf chlorophyll concentration, as measured by organic extraction and spectrophotometric analysis, with output (M) of the Minolta SPAD-502 leaf chlorophyll meter. The relationship is non-linear and can be described by the equation chlorophyll (μmol m−2)=10(M0.265), r2=0.94. Use of such an exponential equation is theoretically justified and forces a more appropriate fit to a limited data set than polynomial equations. The exact relationship will vary from meter to meter, but will be similar and can be readily determined by empirical methods. The ability to rapidly determine leaf chlorophyll concentrations by use of the calibration method reported herein should be useful in studies on photosynthesis and crop physiology.

PLANT CHLOROPHYLL-PROTEIN COMPLEXES: RECENT ADVANCES

This article will review papers that have recently appeared on plant chlorophyll-protein complexes. Because of space limitations, it cannot be exhaustive or critical. We emphasize data that have appeared since the reviews of Anderson [8], Thornber [126], Thornber et al. [130], and Boardman et al. [31], but do not completely exclude some ealier pertinent results. Other recent reviews [35, 114, 127,213 and symposia (Brookhaven Symposium in Biology, Vol. 28, 1976 and ClBA Symposium, New Series 61, 1978) tangentially consider this topic. We will not use the term ‘chlorophyll protein’ in this review, since this term has been used ambiguously by researchers and sometimes misinterpreted to mean either the holocomplex or the apoprotein. Instead, we will use an old term, chlorophyllin, to refer to a holocomplex. No universally accepted nomenclature for chlorophyllins has been established. This and the fact that each group discovering what they believe to be a new chlorophyllin, generally gives the component some unique cryptic designation make it difficult for a reader to realize that 2 different laboratories may be describing the same chlorophyllin. Difficulty also arises because these components, solubilized by detergent solutions, are more difficult to purify than are water-soluble proteins, and hence the quoted characteristics of a given chlorophyllin can vary. Furthermore, most of them so far do not exhibit a distinguishing characteristic that readily permits unequivocal identification of the chlorophyllin. Absorption and emission spectra are not only very similar for each plant chlorophyllin but also the exact position of wavelength maxima for a chlorophyllin varies with the extent of denaturation of that chlorophyllin. In addition, techniques are frequently not identical between different laboratories for determining another frequently quoted characteristic, apparent molecular sizes of chlorophyllins and apoproteins (if the latter can be identified as such following denaturation of the complex). Confusion can also arise because some groups use the same designation for both a specific chlorophyllin and for a larger complex in which this chlorophyllin may be the principal, but not always the only, chlorophyllin present. It would be valuable if a session at the next International Congress on Photosynthesis in 1980 could be organized to establish a generally acceptable nomenclature. The problem grows increasingly acute in view of the large number of apparently different chlorophyllins now being reported,

Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h 3

Background: The identification of IgE epitopes for proteins is the first step in understanding the interaction of allergens with the immune system. Proteins from the legume family have shown in vitro cross-reactivity in IgE-binding assays, but this cross-reactivity is rarely clinically significant. Resolution of this discrepancy requires IgE epitope mapping of legume family protein allergens. Methods: We constructed six fusion proteins representing overlapping regions of soybean glycinin G1 acidic chain. These fusion proteins were used in immunoblotting and a novel sandwich ELISA with pooled sera from soy-allergic individuals to reveal a common IgE-binding region. This region was the focus for IgE epitope mapping using overlapping synthetic peptides. Results: Data from the fusion protein experiments revealed an IgE-binding region consisting of residues F192–I265. Analysis of the overlapping synthetic peptides to this region indicated that IgE epitopes to glycinin G1 acidic chain consist of residues G217–V235 and G253–I265. The epitopes identified for glycinin G1 acidic chain are homologous to IgE epitopes previously identified for the peanut allergen Ara h 3 [1]. However, residues identified by alanine scanning in the peanut epitopes as being important for IgE binding are different in the natural soybean epitopes. Conclusions: The IgE epitopes identified for glycinin G1 acidic chain apparently represent an allergenic region of several legume family seed storage proteins. Our findings indicate that the identification of IgE epitopes and structural analysis of legume family proteins will provide valuable information to the study of food allergies.

Assays for Determination of Protein Concentration

Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high‐throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS‐PAGE sample buffer that is used for equal loading of SDS‐PAGE gels, which is reliable, inexpensive, and quick.

Identification of IgE-Binding Proteins in Soy Lecithin

Background: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. Methods: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. Results: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. Conclusions: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.

Broken Links: The Ephemeral Nature of Educational WWW Hyperlinks

The use of distributed (Internet) resources to enhance both traditional and distance education has caused much excitement in the science education community. However, one of the difficulties with relying on such freely available distributed resources has been the lack of certainty that the resources will be available for students next month, next semester, or next year. We have recently been involved in the development of three graduate-level biochemistry courses designed for high school teachers. Development of these courses relied heavily upon distributed science education resources. As a consequence, they represented a set of authentic science education resources that could be monitored over time to determine their rate of extinction. In total, the three courses contained 515 nonredundant URLs representing either scientific content of science education pedagogy. These have been monitored on a monthly basis during the 14 months since the creation of the courses (August 2000). During this period 85 (16.5%) of the URLs have ceased to function or had their content changed. The most attrition was seen in URLs with the “edu,” “com,” and “org” domain names, in which 17.5, 16.4, and 11% have already become inaccessible.

Aspergillus niger mutants with increased glucose oxidase production

SummaryAspergillus niger NRRL-3, an organism used for the industrial scale production of d-gluconic acid and glucose oxidase (EC 1.1.3.4), was subjected to mutagenesis and selection for acid production on diagnostic media containing methyl red. The plates contained 0.1 M d-glucose, a concentration that does not produce a color change in the medium surrounding mycelia of the parental strain under the conditions employed. Mutagenized spores yielded occasional colonies which were able to grow rapidly and were surrounded by a reddish zone. A number of such presumptive mutants were selected and isolated. Twenty-six such strains were grown in shaken cultures with liquid media containing 0.01, 0.1 or 0.5 M d-glucose, harvested, disrupted and the specific activity of d-glucose oxidase determined. Seven of the mutant strains had glucose oxidase specific activities markedly higher than the parental strain.

Identification and analysis of a conserved immunoglobulin E-binding epitope in soybean G1a and G2a and peanut Ara h 3 glycinins.

To identify conserved immunoglobulin E (IgE)-binding epitopes among legume glycinins, we utilized recombinant soybean G2a and G2a-derived polypeptide fragments. All of these fusion polypeptides bound IgE, and the C-terminal 94-residue fragment appeared to bind more IgE. Using synthetic peptides we identified S219-N233 (S(219)GFAPEFLKEAFGVN(233)) as the dominant IgE-binding epitope. Alanine scanning of this epitope indicated that six amino acids (E224, F225, L226, F230, G231, and V232) contributed most to IgE binding. Among these amino acids, only G231 of soybean G2a is not conserved in soybean G1a (S234) and peanut Ara h 3 (Q256). Synthetic peptides corresponding to the equivalent regions in G1a and Ara h 3 bound IgE in the order Ara h 3>/=soybean G2a>soybean G1a. This sequence represents a new IgE-binding epitope that occurs in a highly conserved region present in legume glycinins. Such IgE-binding sites could provide a molecular explanation for the IgE cross-reactivity observed between soybean and peanut proteins.

Formate dehydrogenase in Arabidopsis thaliana: characterization and possible targeting to the chloroplast

Formate dehydrogenase (E.C. 1.2.1.2) is a mitochondrial-localized NAD-requiring enzyme in green plants. The enzyme activity and corresponding mRNA in leaves of Arabidopsis thaliana are induced by treatment with one-carbon metabolites. The cDNA for the Arabidopsis formate dehydrogenase is similar to that of other plants except for the N-terminal region, which is predicted to target chloroplasts as well as mitochondria. The specific of activity of the enzyme in isolated chloroplasts suggests it is targeted to both mitochondria and chloroplasts in Arabidopsis. Formate dehydrogenase from Arabidopsis was partially purified and K(m) values for formate and NAD(+) were determined to be 10 mM and 65 µM, respectively; the K(i) for NADH was 17 µM. We conclude that formate dehydrogenase is normally present in Arabidopsis chloroplasts and that sensitivity to inhibition by NADH may play a role in whether cellular formate is assimilated or dissimilated.

Reversible denaturation of the soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a beta-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70 degrees C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 degrees C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 degrees C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 degrees C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered beta-sheet proteins.