IntroductionToBindingEquilibrium.docx Page 1 Introduction to Binding Equilibrium Module Specific information about the spectrophotometer below will change depending on the make and model used. Avidin and its natural ligand biotin Avidin is a protein in the oviducts of birds, reptiles and amphibians that is deposited in the whites of their eggs. It binds biotin (vitamin H or B7) with extraordinary affinity (= binding strength) to make the avidin-biotin complex. The binding interaction is non-covalent, meaning that it is mediated by a combination of several individually very weak bonds, rather than by one or more much stronger covalent bonds that are unbreakable under ordinary circumstances. Non-covalent binding, unlike covalent bonding, is reversible: the two interacting molecules can bind to form a complex (the forward reaction), and at the same time the complex can dissociate (come part) to release the two individual molecules again (the reverse reaction). As we ll see, such systems come to a natural equilibrium state, in which the forward and reverse reactions exactly balance. That s true in particular of the avidin-biotin interaction, though in that case dissociation is so slow that actually measuring it is technically challenging. And as if superslow dissociation weren t enough, association (the binding of free biotin to avidin) is super-fast! Superfast association plus super-slow dissociation (unbinding) = a super-super-high affinity. Non-covalent binding interactions are a core feature of biological systems, both inside and outside cells. Paradigmatic examples are the interaction between an antibody and an antigen, or between a cellular receptor and its natural ligand (the hormone or other biomolecule that naturally binds that receptor). For instance, the natural ligand for the insulin receptor on muscle, liver and adipose cells is the hormone insulin in the blood. When insulin engages the insulin receptor, the cell is stimulated to import glucose from the surrounding fluid, thus lowering the blood glucose level. Why did avidin evolve? The answer isn t known definitively, but a good guess is that it acts as an antibiotic to protect the oviduct and egg from microbial contamination. Many microorganisms require biotin as a micronutrient (as do we mammals); and if such a microbe finds itself in an egg white, it s SOL, since any biotin present will be complexed super-tightly to avidin and thus unavailable for the microbe s use. Avidin is a homotetramer, meaning that it consists of four identical subunits. Each subunit is a polypeptide of 128 amino acids with a 10-sugar branched-chain carbohydrate attached to one of its amino acid side chains. Each subunit binds biotin independently of the other subunits; as far as binding equilibrium is concerned, therefore, each tetramer acts the same as would four separate monomers. Consequently, we will calculate avidin concentration in terms of individual monomer subunits. The image below depicts one of the four subunits of avidin, in a cartoon format that traces only the main backbone of the polypeptide chain. The biotin bound to the subunit is rendered in space-filling format. As is evident in the figure, the biotin is buried in a deep pocket in the three-dimensional structure and locked in place by a loop (or flap ) that partially covers it. The chemical environment inside this binding pocket is relatively hydrophobic (= oily); so one consequence of binding is that the biotin has been transferred from the aqueous (watery) bulk solvent to a new hydrophobic organic solvent inside the protein structure.
IntroductionToBindingEquilibrium.docx Page 2 Avidin also binds HABA HABA [(2-(4-hydroxyphenylazo)benzoic acid] is a brightly colored azo dye. As you can see from the structures below, HABA and biotin are very different compounds. HABA Biotin HABA is sparingly soluble in water but very soluble in hydrophobic organic solvents. When a protein with a hydrophobic binding pocket is dissolved in a solution of HABA, therefore, the HABA will sometimes partition from the bulk aqueous solution into the protein s binding pocket (if it can fit). Such is the case with avidin, HABA inhabiting the same hydrophobic binding pocket as does biotin (despite the dissimilarity of their structures). This binding event is easy to detect: whereas HABA dissolved in aqueous solution absorbs light in the near UV ( max = 348 nm) and has a pale yellow color, HABA dissolved in organic solvent including in the binding pocket of avidin absorbs light in the visible range ( max = 500 nm) and has a bright red color. It s this color change that we ll use in the lab to quantify reversible binding of HABA to avidin.
IntroductionToBindingEquilibrium.docx Page 3 Although HABA binds avidin in the same binding pocket as does biotin, the affinities of the two ligands are dramatically different. HABA not only binds avidin much more slowly, it also dissociates from avidin much more rapidly. Unlike the super-strong binding of biotin to avidin, the weak binding of HABA to avidin is easy to quantify and an ideal experimental model for introducing new biology students to binding interactions. Instructions for binding equilibrium experiment Review of pipetting instructions General o In order to maintain accuracy, use the smallest size pipetter that accommodates the volume to be transferred. If you need to transfer 57 µl, for example, use the 100- or 200-µL pipetter, not the 1000-µL pipetter. o Push and release the plunger gradually, especially when aspirating (drawing up the sample; see below). o When there s any liquid in the pipette tip, be sure to hold the pipetter vertically and rightside up, lest the liquid drain back into the working of the pipetter. Contaminated pipetters are tedious to clean. Aspiration from the source vessel (drawing up the dialed volume of liquid) o Put a fresh tip on the pipetter; push the plunger down to the first stop, and keep the plunger at that position; be sure not to push the plunger beyond the stop, which will result in drawing up more than the dialed volume of liquid. o With the plunger depressed to the first stop, immerse the tip of the pipette tip in the source liquid; gradually release the plunger to draw up the dialed volume of liquid; make sure the tip of the pipette tip is in the source liquid throughout aspiration otherwise, you ll aspirate air instead of liquid. o When aspirating small volumes, it s best to just barely submerge the tip of the pipette tip in the source liquid; that minimizes inaccuracy due to liquid sticking to the outside of the pipette tip. o It s particularly important to release the plunger gradually: allowing the plunger to pop up will often aspirate air as well as liquid, leading to extremely large errors. Allowing the plunger to pop up can also project droplets of the liquid into the inner working of the pipetter, necessitating a very tedious cleaning operation. Delivery into the destination vessel o Don t deliver the liquid into air!! The entire volume will rarely drop cleanly into the destination vessel; this is especially true of very small volumes such as 10 µl. o INSTEAD: If the destination vessel is empty, press the tip of the pipette tip gently against the inner wall close to the bottom, and deliver the sample onto the wall so that it flows down the wall to the bottom. If the destination vessel already has liquid in it, immerse the tip of the pipette tip into that liquid and deliver the contents of the pipette tip directly into that liquid. o Press the plunger gradually to the first stop and on to the second stop; keep the plunger depressed to the second stop and withdraw the tip from the destination vessel before releasing the plunger.
IntroductionToBindingEquilibrium.docx Page 4 Discard used tips into the discard beaker Use of Jenway 6705 spectrophotometer 1 You re going to make color measurements with an instrument called a spectrophotometer in particular a Jenway 6705. As soon as you arrive in the lab, we ll turn on the spectrophotometer and allow it to warm up. We ll press the photometrics button (for measurements at a single wavelength) and adjust the wavelength to 500 nm, where the HABA-avidin complex absorbs maximally. The digital readout shows both percent transmittance and absorbance A: we ll use the latter. The relationship between the two metrics is Percent transmittance = 100 10 -A. Thus percent transmittance = 0 corresponds to A = and percent transmittance = 100 corresponds to A = 0. Supplies Each student will be supplied with: Four plastic 1.5-mL microtubes, each containing 1.2 ml of the same HABA solution in DPBS, a buffer at neutral ph; the HABA concentrations in different students tubes will range from 1 to 100 µm Four plastic spectrophotometer cuvettes; below you ll transfer 1000 µl from each microtube into a corresponding cuvette A 500-µL microtube containing water A 500-µL microtube containing avidin in water A 500-µL microtube containing biotin in DPBS Each pair of students will be supplied with: A 100- or 200-µL pipetter and tips (need not be sterile) A 1000-µL pipetter and tips (need not be sterile) 1 Detailed instructions in this subsection will depend on the make and model of the spectrophotometer.
IntroductionToBindingEquilibrium.docx Page 5 A discard beaker labeled Unwanted Materials (for discarding used disposable labware) The class as a whole will use a single spectrophotometer. Students readings will be recorded in a data sheet next to the spectrophotometer. HABA-avidin binding reactions Working as accurately as possible, pipette exactly 40 µl of the avidin solution into two of your 1.5-mL tubes of HABA; mark these tubes (which we ll call the avidin tubes) to distinguish them from the other two 1.5-mL microtubes. Similarly, pipette exactly 40 µl water into both of your other two 1.5-mL tubes (the reference tubes), leaving them unmarked. Close caps and vortex all four tubes. Use the 1000-µL pipetter to transfer exactly 1000 µl from each 1.5-mL microtube into a correspondingly marked cuvette (two avidin cuvettes, two reference cuvettes), discarding both tips and microtubes into the discard beaker; color develops almost immediately. Take cuvettes to the spectrophotometer; read the absorbance at 500 nm twice as follows: Put one of the reference cuvettes in the holder and press the ZERO button Put one of the avidin cuvettes in the holder and press the READ button; record the absorbance in the First read column of the data sheet provided Repeat the previous two substeps with the other reference and avidin cuvettes, recording the absorbance in the Second read column Discard one pair of cuvettes (one avidin, one reference); take the other pair back to your bench for use below. Class discussion questions (before adding avidin or water) 1. What color change will take place when you add avidin to the avidin tubes, and water to the reference tubes? Why? 2. Will the color changes be the same or different for the different students? Explain your answer. Displacement of HABA by biotin Carefully pipette 40 µl of the biotin solution into each cuvette (one reference, one avidin); the amount of biotin added is a slight molar excess over the amount of avidin already in the tube. Use the pipette tip to mix the biotin solution in with the content of the cuvette. Note any color change. If time permits, measure the absorbance at 500 nm as before, recording the absorbance in the After biotin column of the data sheet (otherwise, the instructor will make these measurements after lab). More class discussion questions (before adding biotin) 3. What color change will take place when you add biotin to both the avidin and reference tubes? Why? 4. Will the color changes be the same or different for the different students? Explain your answer.