P39, a novel soybean protein allergen, belongs to a plant-specific protein family and is present in protein storage vacuoles.

Soybean lecithins are seeing increasing use in industry as an emulsifier and food additive. They are also a growing source of human food allergies, which arise principally from the proteins fractionating with the lecithin fraction during manufacture. A previous study (Gu, X.; Beardslee, T.; Zeece, M.; Sarath, G.; Markwwell, J. Int Arch. Allergy Immunol. 2001, 126, 218-225) identified several allergenic proteins in soybean lecithins and a soybean IgE-binding protein termed P39 was discovered. However, very little was known about this protein except that it was coded by the soybean genome. This paper investigates key biological and immunological properties of this potential soybean lecithin allergen. P39 is encoded by a multigene family in soybeans and in several other higher plants. The soybean P39-1 protein and its essentially indistinguishable homologue, P39-2, have been cloned and studied. These proteins and their homologues belong to a family of plant-specific proteins of unknown function. In soybeans, P39-1 is seed specific, and its transcript levels are highest in developing seeds and decline during seed maturation. In contrast, P39 protein was detectable only in the fully mature, dry seed. Subcellular fractionation revealed that P39 protein was strongly associated with oil bodies; however, immunolocalization indicated P39 was distributed in the matrix of the protein storage vacuoles, suggesting that association with oil bodies was an artifact arising from the extraction procedure. By the use of recombinant techniques it has also been documented that IgE-binding epitopes are present on several different portions of the P39-1 polypeptide.

Use of a laboratory exercise on molar absorptivity to help students understand the authority of the primary literature.

To promote understanding of the authority of the primary literature in students taking our biochemistry laboratory courses, a biochemistry laboratory exercise on the determination of an acceptable molar absorptivity value of 2-nitrophenol (2-NP) was developed. This made the laboratory course much more relevant by linking to a thematic thread, ß-galactosidase, that scaffolds concepts in one exercise with those in later exercises. The substrate for the continuous assay of ß-galactosidase is the chromogenic 2-nitrophenyl-ß-D-galactopyranoside that produces 2-NP. In an early laboratory exercise, students determine the wavelength of maximum absorption for the protonated and deprotonated form of 2-NP at various pH values and then determine the molar absorptivity of 2-NP. Students were encouraged to discuss apparent discrepancies not only in their own determinations of molar absorptivity values for 2-NP, but also in the published molar absorptivity values for 2-NP (2,150-21,300 M(-1) cm(-1) ) at almost the same pH and at 420 nm. Finally, the students were led to a publication that serves as an authentic source for molar absorptivity of 2-NP.

Cognitive development and the complexities of the undergraduate learner in the science classroom*.

Students' reactions to classroom learning and the mastery of science vary along a wide spectrum of attitudes and emotions. In particular, we argue here that how learners encounter and learn subject matter is a function of their level of cognitive development. We describe the stages of cognitive development based on the work of William Perry and demonstrate their relevance to the undergraduate science classroom. With examples drawn from biochemistry, we attempt to show that, depending on the student's developmental level, there will be different abilities to handle the range of assignments and activities s/he can expect to experience in the average classroom. The college science instructor can benefit from knowledge of these stages and can work through their implications to develop strategies and techniques to regulate collective student learning.

A mechanistic foundation for instructor-regulated collective learning.

The theory of memory and the ability to influence learning with conscious and unconscious factors, both internal and external, is reviewed. From a connectionist view of memory, the interactive compensatory model of learning is introduced, with an emphasis on the plastic components (motivation, prior knowledge, and metacognition) that can be regulated. Expert students, but not naive students, have a considerable ability to self-regulate their learning processes and improve performance in undergraduate courses. We propose that in a class situation, there are emergent opportunities for an instructor who understands the dynamics of the learning environment to provide regulation for collective learning of multiple students and improve group learning. There are three interfaces for instructor-regulated collective learning, and examples are provided for each to suggest how instructors may effectively provide regulation.

Stability of the allergenic soybean Kunitz trypsin inhibitor.

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.

Kinetic behavior of the Arabidopsis thaliana leaf formate dehydrogenase is thermally sensitive.

Two previous kinetic studies on the Arabidopsis thaliana leaf NAD-dependent formate dehydrogenase (EC 1.2.1.2) have demonstrated two very different sets of Km values for the formate and NAD+ substrates. We examined the kinetics of the enzyme partially purified from a leaf extract by gel-filtration desalting and chromatography on DEAE-cellulose, as well as by isolation of a mitochondria-enriched fraction obtained by differential centrifugation. Both of these methods produce a formate dehydrogenase enzyme with the higher Km values of approximately 10 mmol/L formate and 75 mumol/L NAD+. The kinetic properties of the Arabidopsis formate dehydrogenase expressed to high levels in transgenic tobacco plants were also those of the high Km form. The high Km form of the enzyme converted to a low Km form by heating for 5 minutes at 60 degrees C. An Arrhenius plot of the activity during the heating process was linear, indicating that the heating did not cause alterations in either the active site or the thermal dependence of the catalytic reaction. We conclude that the native form of the formate dehydrogenase probably resembles the form with the higher Km values. Heating seemingly converts this native enzyme to the molten globule state and cooling results in formation of a non-native structure with altered kinetic properties.

The human side of science education: Using McGregor's theory Y as a framework for improving student motivation*.

Student motivation is correlated with learning. Douglas McGregor's Theory X and Theory Y as a basis for understanding and improving motivation in the business world can be directly applied to the science classroom. Teachers with a Theory Y perspective (students naturally want to learn) provide increased motivation for students and promote more active learning than Theory X-style teachers who do not view students as active learners. Many teachers are not aware of their Theory X/Theory Y orientation and how this bias may be impacting their interaction with students. This article explores the benefits of moving from a Theory X to a more Theory Y style of teaching.

Facilitating student understanding of buffering by an integration of mathematics and chemical concepts.

We describe a simple undergraduate exercise involving the titration of a weak acid by a strong base using a pH meter and a micropipette. Students then use their data and carry out graphical analyses with a spreadsheet. The analyses involve using mathematical concepts such as first-derivative and semi-log plots and provide an opportunity for collaboration between biochemistry and mathematics instructors. By focusing on titration data, rather than the titration process, and using a variety of graphical transformations, we believe that students achieve a deeper understanding of the concept of buffering.

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.

Assays for determination of protein concentration.

Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix 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.

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.

C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen.

Gly m Bd 28 K is a major soybean (Glycine max Merr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the beta subunit of soybean beta-conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first beta-sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans.

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.