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Transcript
First portion: A chromophorogenic assay Non-absorbing compounds can be detected via a reaction that generates a chromophore in proportion to the compound’s concentration. ! Either a known ! or a standard curve are used to relate the A to the starting compound’s concentration. (The standard curve in-essence yields a !). ! We will use the Bradford reagent, which is a solution of Coomassie blue G250 in ethanol/phosphoric acid. This is less tricky than the text’s recommendation of FolinCiocalteau. ! The product sheet for Sigma’s Bradford reagent is provided on the course web site. We will use assay 2 type ‘A’ from that sheet. ! BIOCHEMISTRY LAB CHE-554 Experiment #1 Spectrophotometry Professor Testa Making standard samples -1 Precautions for Chromophorogenic assays ! ! ! ! ! 3 The reaction must be limited ONLY by the compound to be measured. (Every molecule of compound is counted) A linear relationship must be demonstrated for the absorbance and reaction that forms the dye. Conduct the experiment in such a way that the readings corresponding to unknown samples fall within the reading that make up the standard curve. If necessary, make dilutions of the unknown. Do this BEFORE conduction the reaction. ! ! The active ingredient is Coomassie blue G250, which binds primarily to arginine residues. The sigma reagent is used as follows: 3.0 ml reagent + 0.1 ml solution to be tested. First generate a series of known concentrations of the standard protein solution as follows: ! 4 Vol. of Protein stdd. soln. (µl) Vol. of buffer (µl) 100 0.0 80 20 60 40 40 60 20 80 0 100 Reading the standard samples Making standard samples -2 ! ! ! 5 In your notebook, be sure that you have a name for each sample included in your table and that this name is used to label the corresponding tube. Make these samples up in test tubes that have sufficient capacity for the addition of 3 ml of the Bradford reagent and subsequent mixing without spilling. Add 3 ml Bradford reagent to each tube. DO NOT dip a pipette tip into the master bottle, pour a little reagent out of the bottle and dispense from this aliquot. If there is left-over and it is not contaminated, pass it along to a colleague for use. ! ! 6 Mix each sample and then wait at least 5 min. but no longer than 45 min. (Observe changes in your samples as a function of time, and take photographs.) Read A595 and record these results. Making the unknown samples ! ! Adapt the protocol detailed above to make a series of five samples of the unknown, plus a ‘null’ sample. React these with Bradford reagent as above and read them as above. Questions ! ! ! ! ! 7 8 Why do we test a whole series of standard samples instead of just one ? Why do we include a null sample ? Why do we test a whole series of dilutions of our unknown sample instead of just one ? What is a potential pitfall of making a standard curve from one species of protein as a basis for determining the concentration of a different protein ? What are potential hazards of working with the Bradford reagent and why ? Second Portion: Direct absorbance measurement on a protein ! ! ! 9 We will exploit the strong absorbance of UV radiation by tryptophan (Trp) and tyrosine (Tyr) side chains in a protein. Each protein species has a characteristic 3D structure that places its various Trp and Tyr side chains in unique environments and causes them to have extinction coefficients that vary quite widely. However if a protein is denatured to a ‘random coil’ all the side chains are exposed to the medium and behave as if they were all simply amino acids dissolved in that medium. Denatured protein ! ! In a denaturing medium, the extinction coefficient of the protein at 280 nm can be approximated as the sum of the contributions of the Trps and the Tyrs: !protein = nTrp • !Trp + nTyr • !Tyr We will use the protein lysozyme from chicken egg white. the amino acid sequence of this protein is known1 : LYS ARG ASN THR TYR ASP PRO VAL MET THR ! VAL HIS TRP GLN GLY GLY CYS ASN ASN ASP PHE GLY VAL ALA ILE ARG SER CYS ALA VAL GLY LEU CYS THR LEU THR ALA ALA TRP GLN ARG ASP ALA ASN GLN PRO LEU LYS VAL ALA CYS ASN ALA ARG ILE GLY LEU LYS ALA TRP GLU TYR LYS ASN ASN SER SER ILE TRP ILE LEU ARG PHE THR SER ARG SER VAL ARG ARG ALA GLY GLU ASP ARG ASN ASP SER ASN GLY ALA TYR SER GLY TRP LEU ILE ASP ARG CYS ALA SER ASN SER TRP CYS THR GLY CYS ARG MET LEU PHE THR CYS ASN ALA ASN LYS LEU LYS GLY ASN ASP ASN ILE SER GLY GLY In our denaturing medium, at 280 nm !Trp = 5690 M-1cm-1 and !Tyr = 1280 M-1cm-1. 10 1 Canfield (1963) ‘The Amino Acid Sequence of Egg White Lysozyme‘ Chem. 238:2698-707 J Biol Questions to be answered in your theory section and pre-lab. ! ! ! ! 11 What is the extinction coefficient of denatured lysozyme at 280 nm in our denaturing medium ? (Show your work.) What potential hazards are associated with working with guanidinium HCl ? Does this material contain HCl ? Look at the MSDS for guanidinium HCl available on the course web site and assess this material’s toxicity by comparing its LD50 with those of glucose and aspiring, which are also posted. The experiment ! ! ! ! 12 The implication is that under denaturing conditions, we can determine the concentration of any protein if we know its Trp and Tyr content. We will use this strategy to determine the concentration of a lysozyme solution. This denatured solution will be derived from a native (folded) solution of lysozyme by a known dilution. Thus you will calculate the concentration of the parent native solution. The calculated concentration and the measured absorbance at 280 will then be used to calculate the native protein’s extinction coefficient at 280 nm. Protocol, continued Protocol ! ! ! ! 13 Measure the A280 of a solution of native (folded) lysozyme provided by your T.A. (This will be in 20 mM phosphate buffer at pH 6.5). Use a quartz cuvetted for this measurement. Recover the native lysozyme from the cuvette. Dilute 2.5 ml of the native lysozyme with 7.5 ml of 8 M guanidinium hydrochloride in the same phosphate buffer. What will be the final concentration of guanidinium hydrochloride ? By what factor will the denatured lysozyme concentration be related to the original concentration of native lysozyme ? ! ! ! ! 14 As a pedagogical exercise, we will pretend we only have one quartz cuvette each. Thus you will need to re-use the cuvette you used for the native lysozyme sample. Transfer 3 ml of denatured lysozyme to the cuvette and measure its A280 (first measurement). Discard this solution and transfer a second 3 ml aliquot of denatured lysozyme to the cuvette and measure its A280 (second measurement). Discard this solution too and transfer a second 3 ml aliquot of denatured lysozyme to the cuvette and measure its A280 (third measurement). Post-lab thoughts Questions ! ! ! ! How do your three readings for the A280 of denatured lysozyme compare ? Should you use the average of these values ? What value should you use and how will you know if it is valid ? Use your calculated extinction coefficient for denatured lysozyme to calculate the concentration of denatured lysozyme. ! Now calculate the lysozyme concentration of the original native solution of lysozyme. ! Use this and the A280 you measured for native lysozyme to calculate the extinction coefficient of native 15 lysozyme. ! ! ! 16 How does the extinction coefficient you determined for native lysozyme compare with published values ? (look up at least one, and provide your source. Remember that lysozyme from different sources can have different extinction coefficients, you are looking for hen egg white lysozyme. Hen is also known as Galus galus. Compare your measured absorbances for the unknown with those measured by a classmate (say who). Discuss the magnitude and possibles causes of any differences. Also answer questions 2, 3, 4 from our text, experiment 1(page 24).