That accuracy, in conjunction with the well-established physical principles from which the mathematical model was developed mass action and Beer's law , give us considerable confidence in Equation 3 as an explanation of the hidden physical processes underlying the observed results. By the same token, we can be relatively confident of predictions based on that equation.
Those predictions are extensive. The model allows the equilibrium state to be predicted for different concentrations of avidin and HABA than those actually investigated. Moreover, it is the end-point of an even deeper mathematical model describing the entire time-course along which the system attains equilibrium Figure 4. The pedagogical approach of the equilibrium binding lab module is detailed on the companion website, and will be summarized only briefly here.
Inquiry was focused instead on the mental process of developing scientific understanding from the data, from relevant scientific principles presented in reading and lectures e. First, the inquiry evolved in planned stages, each class session and homework task focusing on a restricted part of the overall inquiry that students could master for themselves.
Second, short lectures and readings introduced specific scientific principles e. Third, prior modules provided worked examples that could be applied to the problem at hand. In particular, the Beer's law module not only introduced students to spectrophotometry and to the law itself, but also included a computer lab in which least-squares optimization was used to estimate an absorption coefficient parameter; at the same time, that computer lab served to enhance their mastery of the spreadsheet program.
In spite of extensive guidance, there was a great deal of intellectual distance for students themselves to traverse, both individually and as a group. For instance, they had to recognize that their prior module on Beer's law taught them not just about the absorption coefficient for the particular chromophore in that module, but about the absorption coefficients for chromophores in general, including the avidin-HABA complex; and not just about estimating absorption coefficients, but about estimating parameters in general, of any kind and in any number.
Such generalizations, which come so naturally to experienced scientists, and which are so central to scientific practice, are a revelation to beginning science students. As central as mathematics is to the practice of science, and was to MLS's ambitions, the MLS course did not emphasize new mathematical techniques, as in traditional modeling courses.
We sought instead to show students how even the elementary mathematics they had already learned in high school illuminates a great diversity of biological phenomena. Pettey Principal Investigator. Detailed instructions are available in the BindingEquilibriumPreparation document on the companion website.
Measure A fine yellow precipitate forms temporarily but disperses immediately. Continue vortexing until the precipitate is fully dissolved. Upon thawing and before opening a tube, mix to ensure homogeneity and centrifuge briefly at low speed to drive all liquid to the bottom. Biotin stock solution : The mM biotin stock solution is used to titrate avidin second bullet in the next section ; therefore biotin solid of high purity should be used e.
Avidin stock solution. Avidin can be purchased in or mg quantities Merck-Millipore , enough for one and ten student labs, respectively; the smaller amount is about twice as expensive per mg as the larger amount. Here I describe the stock solution for the smaller amount, to be used within a few days without freezing. The BindingEquilibriumPreparation document on the companion website describes preparing frozen aliquots of the larger amount, to be used over a period of years.
The entire contents of the vial nominally 10 mg is dissolved in 1. The supernatant is transferred to a larger vessel capacity at least 3 mL and diluted with 1 mL additional water total volume 2. The nominal avidin subunit concentration is Preparations just before lab : The BindingEquilibriumPreparationWorksheet Microsoft Excel spreadsheet on the companion website automates the calculations below.
Starting with the mM biotin stock above, make at least 2. Thaw the mL tube of HABA stock solution above, vortex the tube vigorously, and centrifuge it briefly to drive solution to bottom. It is significantly more accurate to measure the DPBS diluent into the tube by weight rather than by volume. The actual HABA concentrations can differ slightly from the target concentrations if convenient, and the volumes can differ slightly from 5.
Dispense exactly 1. Recipient s will receive an email with a link to 'Understanding Reversible Molecular Binding' and will not need an account to access the content. Sign In or Create an Account. User Tools. Sign In. Skip Nav Destination Article Navigation.
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Parameter Estimation. Pedagogical Strategy. Article Navigation. Research Article November 01 Smith George P. This Site. Google Scholar. The American Biology Teacher 79 9 : — Get Permissions. Cite Icon Cite. Figure 1. View large Download slide. Three-dimensional structure of biotin bound to an avidin subunit Livnah et al.
The avidin is rendered in a format that traces only the main backbone of the polypeptide chain. The biotin is rendered in space-filling format. Figure 2. Results of the binding equilibrium experiment for a recent semester. The x axis plots the total input concentration of HABA molecules, regardless of whether or not they are bound to an avidin partner.
Filled triangles and open circles represent the duplicate absorbance measurements for each student. The open squares represent the absorbance measurements after adding biotin. The dashed line marks 0 absorbance. The solid curve and the best-fitting K D will be explained in the section on Parameter Estimation.
Figure 3. The upper and lower expressions represent the forward association and reverse dissociation reactions, respectively. Figure 4.
Time course of the binding reaction. Green, N. A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Hmelo-Silver, C. Scaffolding and achievement in problem-based and inquiry learning: A response to Kirschner, Sweller, and Clark Kirschner, P. Why minimal guidance during instruction does not work: An analysis of the failure of constructivist, discovery, problem-based, experiential, and inquiry-based teaching.
Livnah, O. National Research Council. Ninfa, A. Smith, G. White, H. Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs: Discovery of a new biotin-binding protein. HABA stock solution:. All rights reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press's Reprints and Permissions web page, www.
Send Email Recipient s will receive an email with a link to 'Understanding Reversible Molecular Binding' and will not need an account to access the content. Recipient Subject: Understanding Reversible Molecular Binding.
Optional Message: Optional message may have a maximum of characters. Citing articles via Web Of Science 1. Email alerts Article Activity Alert. Latest Issue Alert. Stay Informed Sign up for eNews. Courses Browse All Courses. Because this central 2,3-bisphosphoglycerate—binding cavity of deoxyHb narrows considerably upon O 2 binding, oxyHb exhibits significantly reduced affinity for band 3; that is, creating a prominent O 2 -regulated protein-protein interaction in the red cell that switches on and off precisely over the range of O 2 concentrations that are experienced by the erythrocyte in vivo.
The proximity of the deoxyHb-binding site on band 3 to the docking sites of glycolytic enzymes on band 3 can explain the O 2 -mediated change in glucose flux between glycolysis and the pentose phosphate pathway. Because the glycolytic enzymes are inhibited when bound to band 3, 54 glucose flux is shifted toward the pentose phosphate pathway under oxygenated conditions.
In contrast, when the cell is deoxygenated and deoxyHb displaces the GEs, glucose can be more aggressively consumed by glycolysis. We propose that deoxyHb will sterically displace ankyrin from band 3 whenever it occupies its binding site on band 3. Further, the probable adaptive advantage of this O 2 -mediated switch is that periods of deoxygenation should lead to rupture of multiple ankyrin-band 3 bridges, leading to increased deformability, allowing more rapid flow of deoxygenated RBCs from the tissues to the lungs.
ATP release through the pannexin-1 channel in RBCs is thought to be O 2 dependent, 59 suggesting that some type of communication between band 3 and pannexin-1 may exist.
In addition, binding of deoxyHb to band 3 is known to induce global conformational changes in band 3 and associated membrane-spanning proteins. The release of ATP into the vasculature upon RBC deoxygenation should promote generation of NO via activation of P2Y receptors on endothelial cells, 46 thereby causing vasodilation and accelerated transit of the hypoxic RBCs to the lungs. In summary, commonly held beliefs that the erythrocyte is an inert cell that passively circulates for days before its removal by macrophages greatly underestimates the sensitive communication between this cell and its environment.
We show here that multiple important RBC properties are exquisitely responsive to the oxygenation state of the cell, allowing the erythrocyte to better perform its gas transport function. The publication costs of this article were defrayed in part by page charge payment.
Contribution: H. Correspondence: Philip S. Sign In or Create an Account. Sign In. Skip Nav Destination Content Menu. Close Key Points. Materials and methods. Article Navigation. Red Cells, Iron, and Erythropoiesis December 8, This Site. Google Scholar. Mary M. McKenna , Mary M. Nathan A.
Krump , Nathan A. Laurel Mendelsohn , Laurel Mendelsohn. Lisa J. Garrett , Lisa J. David M. Bodine , David M. Philip S. Low Philip S. Blood 23 : — Article history Submitted:.
Connected Content. A related article has been published: Mechanism of O 2 -sensitive red cell properties. Cite Icon Cite.
Figure 1. View large Download PPT. Figure 2. Table 1. Transgenic murine hematologic indices. View Large. Figure 3. Figure 4. Figure 5. Figure 6. The online version of this article contains a data supplement.
Conflict-of-interest disclosure: The authors declare no competing financial interests. Erythrocyte signal transduction pathways, their oxygenation dependence and functional significance.
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The reaction between nitrite and deoxyhemoglobin. Reassessment of reaction kinetics and stoichiometry. Supplemental data Document 1. Add comment Close comment form modal. Submit a comment. Comment title. You have entered an invalid code. Submit Cancel. Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion. Please check for further notifications by email. Volume , Issue Previous Article Next Article.
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