Background BIOCHEMISTRY LAB CHE-554. Experiment #1 Spectrophotometry

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1 BIOCHEMISTRY LAB Experiment #1 Spectrophotometry CHE-554 In day 1 we will use spectrophotometry as an analytical technique using a known extinction coefficient to assess the precision and accuracy of common operations in a biological chemistry lab: pipetting. In day 2 we will undertake an experiment wherein we will determine the extinction coefficient of a protein and then use it to learn the concentration in a solution. Relevant material is provided in the text in experiment 1, beginning page 15. However, we will use the Bradford Professor Testa reagent instead of Folin-Ciocalteau, we will omit studies of riboflavin and adenine, we will instead measure A280 of lysozyme protein when native and when denatured. Thus this experiment will have two parts: 1-Bradford reagent chromogenic assay and -2- A280 of lysozyme. (Introductory material beginning on page 3 of the text may also prove useful.) Background! Photometry relates to the study of light.! An experimental tool for producing and measuring a spectrum of light, visible or ultraviolet, is the UV-VIS spectrophotometer.! The UV-VIS spectrophotometer produces incident light and measures the light that passes through the sample (is not absorbed). The machine calculates how much light was absorbed, and presents that to the user.! Solutions absorb at specific wavelengths (energy levels) of light, and this is a function of the material in the solution. Particular materials have a characteristic absorption spectra through a range of wavelengths. Therefore, one can obtain information about a solution by measuring its absorbance.! The absorption of a solution at a specific wavelength also depends on the concentration of sample. Therefore, one can measure the concentration of known material via UV-VIS spectroscopy.! In the visible range, wavelengths of light not absorbed by the sample make up the color of the sample that you see.

2 Theory of absorbance -1 Each photon has a probability γ of being absorbed if it encounters a molecule of dye (absorbing substance). X photons incident (1-γ)X photons transmitted Theory of absorbance -2 # photons absorbed = # photons entering dye x γnaπr 2 C l = # photons entering dye x ζ C l d (# photons) = - # photons x ζ C dl Upon passage through a small amount of solution, the path length is very short: dl (a small change in position l ) dp= -ζ C dl x P dp/p = - ζ C dl The number of photons changes by a small amount: dp. γx photons absorbed (γ = here) r If a photon s path passes through a solution with C x NA molecules of dye per L, we consider that a photon affects molecules within a cross-section of area πr 2 and the length of the path through the dye is l (letter l ), then the photon is expected to encounter C x NA x πr 2 x l molecules and have a probability γ C NA πr 2 x l of being absorbed. l ln(p)-ln(po) = -ζ Cl - - ζ C 0 ln(p/po) = -ζ Cl P/Po = e -ζcl Incident light ln (P/Po) = log(p/po) log(p/po) = -ζ Cl /2.303 = - εcl, ε=ζ /2.303 transmitted light 3 r is assumed to depend only on the molecule s identity and the wavelength of light. C is the concentration (moles L -1 ). # photons absorbed = # photons entering dye x (γnaπr 2 ) C l Po (power at zero thickness of absorber) 4 l (letter l) Pl (power at l thickness of absorber)

3 Theory of absorbance -3 log(io/i) = A = εcl, Eq. 1-7 C is concentration, l is path length, ε is molar extinction coefficient. ε (and therefore A) is a function of the wavelength of the light. If the dye is too concentrated, some molecules may be in the shade of others and not have their expected probability of absorbing a photon. Non-linear regime, Beer-Lambert law no longer holds for high C or long path lengths. Plot A/ Cl = ε The Beer-Lambert Law A = ε c l This equation relates the concentration of the lightabsorbing compound and the path-length of incident light to the absorbance of a solution. A is the measured absorbance of the sample ε is the extinction coefficient, which is a constant that depends on the structure of the material, the wavelength of incident light, and the solvent C is the calculated concentration of the sample l is the length of the path that the incident light travels through the sample A = εcl, slope = εl Units of ε: M -1 cm -1 In these experiments, measure the absorbance using a spectrophotometer and calculate the concentration of sample in solution. 5

4 The electromagnetic spectrum Absorbance vs. wavelength λ (nm) ε (M -1 cm -1 ) nm ! ! in ethanol band II band I

5 Significance of wavelength Transitions between electronic states λ = c/ν, ΔE = h ν, = hc/λ ν is the frequency, λ is the wavelength and c is the speed of light. h is Planck s constant, = 6.6 x J/s λmax is the wavelength with the maximal ε for a given band. It corresponds to the energy of the transition associated with that band. Long wavelength photons carry less energy, shortwavelength photons carry more energy. Longer wavelengths n-π* transitions, mid-wavelenths π-π* transitions. Usually 260, 180 nm, respectively. Eg. N-containing bases of DNA: 260 nm absorbance. Hence the danger of UV light to DNA

6 The visible portion of the EM spectrum Making a measurement Violet: nm Indigo: nm Blue: nm Green: nm Yellow: nm Orange: nm Red: nm Spectrophotometer 11

7 Selecting a wavelength Spectrophotometer Sample standard curve Sample data set Interpolation or the use of the equation of the line allows determination of the unknown concentration. Slit A Sample 14 C (M) A non-zero intercept may be real, for example due to a reaction with the buffer.

8 Sample standard curve Sample standard curve In this case the unknown falls out of range and requires extrapolation, which is much more dangerous than interpolation. In this case the unknown falls out of range and requires extrapolation, which is much more dangerous than interpolation. A A 15 C (M) 16 C (M)

9 17 Why use absorbance?! It is often a MUCH more accurate way to know concentrations than the weights and volumes used to produce them.! The advantage provided depends on the magnitude of the extinction coefficient (why?)! Accuracy is different from precision (how?)! We will compare the actual concentration of a solution prepared by weighing, dissolving and diluting with the concentration predicted based on the execution plan. 18 Validation of techniques and refresher on uncertainties. Bromophenol blue Concentration? c = mass/mw vol Make an illegal measurement, break Beer- Lambert s law and evaluate error. Dilute to A < 1 Concentration? c = A/εl Statistics based on independent repetitions of the dilutions and absorbance measurements. Validation based on comparison with authentic standard solution.

10 First experiment: 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 ε or a standard curve are used to relate the A to the starting compound s concentration. (The standard curve in-essence yields ε).! 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 Folin- Ciocalteau.! The product sheet for Sigma s Bradford reagent is provided on the course web site. We will use a 19 variant of the standard procedure A. 20 Bradford Assay! Marion Bradford published and patented the assay. Bradford, M. M. (1976) Anal. Biochem. 72: (This is one of the most heavily cited scholarly articles of all time).! Based on a shift in the absorbance maximum of Coomassie brilliant blue G-250 upon binding to arginine side chain (red form of dye converted to a more blue form).! Two chemical bases for the λmax shift: Acidic dye is added to protein, λmax of the dye shifts from 465 nm to 595 nm. Dye binds to basic and aromatic amino acids especially Arg. Detergents and alkaline phs interfere with the dye s colour shift.

11 Coomassie brilliant blue G-250! At acidic ph, the Ns are protonated, the sulfonates remain ionized, net charge is +1 colour is red.! At neutral ph the Ns are deprotonated, only one is +ve, molecule is an an anion. Molecule is green with ε ~ 43,000 M -1 cm -1.! Binding to protein stabilizes the anion, and produces the blue-green form even when free dye molecules remain cationic (red).! Initially used to dye wool (keratin). - +Arg + R-250 lacks two methyl groups Precautions for Chromophorogenic assays! 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 the reactant 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 conducting the reaction. Structure of Coomassie brilliant blue 21 G Coomassie_Brilliant_Blue 22

12 Second Experiment: 2- Direct absorbance measurement on a protein! 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 Amino acids that absorb strongly in the UV. Garrett and Grisham, 3rd ed. Fig. 4.15

13 A typical protein: Lysozyme UV-absorbing amino acids 25 2ZYP.pdb 26 6 Trp and 3 Tyr.

14 UV-absorbing amino acids! 6 Trp Trp and and 3 Tyr. Tyr.! Some are buried, others are stacked. 27 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: εprotein = ntrp εtrp + ntyr εtyr! We will use the protein lysozyme from chicken egg white. the amino acid sequence of this protein is known 1 : LYS VAL PHE GLY ARG CYS GLU LEU ALA ALA ALA MET LYS ARG HIS GLY LEU ASP ASN TYR ARG GLY TYR SER LEU GLY ASN TRP VAL CYS ALA ALA LYS PHE GLU SER ASN PHE ASN THR GLN ALA THR ASN ARG ASN THR ASP GLY SER THR ASP TYR GLY ILE LEU GLN ILE ASN SER ARG TRP TRP CYS ASN ASP GLY ARG THR PRO GLY SER ARG ASN LEU CYS ASN ILE PRO CYS SER ALA LEU LEU SER SER ASP ILE THR ALA SER VAL ASN CYS ALA LYS LYS ILE VAL SER ASP GLY ASN GLY MET ASN ALA TRP VAL ALA TRP ARG ASN ARG CYS LYS GLY THR ASP VAL GLN ALA TRP ILE ARG GLY CYS ARG LEU! In our denaturing medium, at 280 nm εtrp = 5690 M -1 cm -1 and εtyr = 1280 M -1 cm J Biol Chem Aug;238:

15 The experiment! We will determine the concentration of a lysozyme solution indirectly, by first determining the concentration of an aliquot of that solution that we dilute into denaturing conditions. We do that because under denaturing conditions, we can calculate the extinction coefficient because we know the Trp and Tyr content. This extinction coefficient enables us to determine the concentration.! From the dilution factor we will calculate the concentration of the parent native solution.! The calculated concentration and the measured absorbance at 280 nm will then be used to calculate the native protein s extinction coefficient at nm. 30 Experiment 1, Precautions for Chromophorogenic assays! 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 the reactant 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 conducting the reaction.

16 The assay conditions! The Bradford concentrate contains methanol and phosphoric acid.! These are potentially hazardous.! How might this formulation be changed for reduced danger?! How will you handle it?! How will you dispose of your reactions?! Standard has a concentration of 2 x 10-2 mm. (Check with your T.A.) 31