BIOCHEMISTRY LAB CHE-554

Spectrophotometry

Professor Testa

Experiment #1

In day 1 we will use spectrophotometry as an analytical technique using a known extinction coefficient to assess the precision and accuracy of a common operation in a biological chemistry lab: pipetting.

In day 2 we will undertake an experiment wherein we will determine the extinction coefficient of a protein thus permitting direct determination of concentration in the future. We will also use a common indirect method wherein a dye is used in a chromogenic assay.Relevant material is provided in the text in experiment 1, beginning page 15. However, we will use the Bradford reagent instead of Folin-Ciocalteau, we will omit studies of riboflavin and adenine. (We will instead demonstrate a direct protein assay.)

(Introductory material beginning on page 3 of the text may also prove useful.)1

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

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Each photon has a probability γ of being absorbed if it encounters a molecule of dye (absorbing substance).

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 πr2 and the length of the path through the dye is l (letter ‘l’), then the photon is expected to encounter C x NA x πr2 x l molecules and have a probability γ C NA πr2 x l of being absorbed.

r is assumed to depend only on the molecule’s identity and the wavelength of light. C is the concentration (moles L-1).

X photons incident

γX photons absorbed

(1-γ)X photons transmitted

(γ = 0.333 here)

r

l

# photons absorbed = # photons entering dye x (γNAπr2) C l

Theory of absorbance -2

4

Incident light

Po (power at zero thickness of absorber)

transmitted light

Pl (power at ‘l’ thickness of absorber)

l (letter l)

# photons absorbed = # photons entering dye x γNAπr2 C l = # photons entering dye x ζ C ld (# photons) = - # photons x ζ C dl

dP= -ζ C dl x PdP/P = - ζ C dl

ln(P)-ln(Po) = -ζ Cl - -ζ C 0ln(P/Po) = -ζ ClP/Po = e-ζCl

log(P/Po) = -ζ Cl /2.303 = - εCl, ε=ζ /2.303

Upon passage through a small amount of solution, the path length is very short: dl (a small change in position ‘l’)

The number of photons changes by a small amount: dP.

ln (P/Po) = 2.303 log(P/Po)

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm

Theory of absorbance -3

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log(Io/I) = A = εCl, C is concentration, l is path length, ε is molar extinction coefficient.

Eq. 1-7

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.

A = εCl, slope = εl

Non-linear regime, Beer-Lambert law no longer holds for high C or long path lengths.

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm

Units of ε: M-1 cm-1

Plot A/ Cl = ε

The Beer-Lambert Law

This equation relates the concentration of the light-absorbing compound and the path-length of incident light to the absorbance of a solution.

A = ε c l

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

In these experiments, measure the absorbance using a spectrophotometer and calculate the concentration of sample in solution.

The electromagnetic spectrum

7 http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Absorbance vs. wavelength

8 http://omlc.ogi.edu/spectra/PhotochemCAD/html/riboflavin.html

in ethanol

λ (nm) ε (M-1 cm-1) 266.50 nm 33000349.00! 11138445.25! 11051

band Iband II

Significance of wavelength

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λ = c/ν, ν is the frequency, λ is the wavelength and c is the speed of light.

ΔE = h ν, h is Planck’s constant, = 6.6 x 10-34J/s = hc/λ

Long wavelength photons carry less energy, short-wavelength photons carry more energy.

λmax is the wavelength with the maximal ε for a given band.It corresponds to the energy of the transition associated with that band.

Transitions between electronic states

10 http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Longer wavelengths ≈ n-π* transitions,mid-wavelenths ≈ π-π* transitions.

In biochemistry 280 - 260 nm, 180 nm, respectively.

Eg. N-containing bases of DNA: 260 nm absorbance.Hence the danger of UV light to DNA.

The visible portion of the EM spectrum

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• Violet:   400 - 420 nm • Indigo:   420 - 440 nm • Blue:   440 - 490 nm • Green:   490 - 570 nm • Yellow:   570 - 585 nm • Orange:   585 - 620 nm • Red:   620 - 780 nm

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Making a measurement

Spectrophotometer

Selecting a wavelength

Spectrophotometer

Slit

Sample

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 ?)

! This is the object of our activities on Day 1.

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Sample standard curveSample data setInterpolation or the use of the equation of the line allows determination of the unknown concentration.

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A

C (M)

A non-zero intercept may be real, for example due to a reaction with the buffer.

Sample standard curve

In this case the unknown falls out of range and requires extrapolation, which is much more dangerous than interpolation.

16C (M)

A

Sample standard curve

In this case the unknown falls out of range and requires extrapolation, which is much more dangerous than interpolation.

17C (M)

ASample standard curve

Determination of the extinction coefficient. A = εC + 0 (with a 1 cm path length)Assuming an intercept ≈ 0, the slope is ε.Why do we use the slope instead of just one (C, A) pair?

18C (M)

A

What we will do

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Day 1: Validation of techniques and refresher on uncertainties.

Bromophenol blue

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Concentration ?c = mass/mw•vol

Concentration ?c = A/εl

Statistics based on independent repetitions of the dilutions and absorbance measurements.

Validation based on comparison with authentic standard solution.

Make an ‘illegal’ measurement,

break Beer-Lambert’s law .

Concentration ? c = A/εl ?

Dilute to A < 1

Day 2, Experiment 1:A chromogenic 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 variant of the standard procedure A. 21

Bradford Assay! Marion Bradford published and

patented the assay. Bradford, M. M. (1976) Anal. Biochem. 72: 248-254. (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.

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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-1cm-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).

23Structure of Coomassie brilliant blue G-250. http://en.wikipedia.org/wiki/Coomassie_Brilliant_Blue

R-250 lacks two methyl groups

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Brilliant Blue G250

Coomassie Brilliant Blue and Mechanisms of Protein Staining and Bradford-Assay

ORIGIN The name Coomassie was first used in the late 19th century, adopted from the town of Coomassie (modern-day

Kumasi in Ghana), as a trade name of the dye manufacturer Levinstein Ltd. for two similar triphenylmethane dyes

used as acid wool dyes. The two blue dyes were then first produced in 1913 by Max Weiler based in Elberfeld,

Germany. Today, the term ‘CoomassieTM’ is a registered trademark of Imperial Chemical Industries.

Overall, there are approx. 40 dyes called ‘CoomassieTM xy’, while only CoomassieTM G250 and CoomassieTM R250

play a crucial role in biochemical analyses. During the last years, however, most authors referred to these dyes

simply as ‘CoomassieTM’ without specifying which dye is actually meant.

The term ‘250’ originally was used for denotation of the purity of the

dye. The suffix ‘G’ in ‘Brilliant Blue G250’ was added to describe the

slightly greenish colour of the blue dye. The suffix ‘R’ in ‘Brilliant Blue

R250’ is an abbreviation for ‘red’ as the blue colour of the dye has a slight reddish

tint. CoomassieTM Brilliant Blue G-250 differs from CoomassieTM Brilliant Blue R-250

by the addition of two methyl groups.

BACKGROUND OF COLOUR CHANGES The colour of the two dyes depends on the acidity of the solution and on its binding status to amino acids or

peptides. At a pH of less than 0 the dye has a red colour with an absorption maximum at a wavelength of 470 nm.

At a pH of around 1 the dye is green with an

absorption maximum at 620 nm while above

pH 2 the dye is bright blue with a maximum at

595 nm.

The different colours result from the differently

charged states of the dye molecule, corresponding to the amount of positive charges at the three nitrogen atoms

present, while the two sulfonic acid groups are normally always negatively charged.

• At a pH of around zero, all three nitrogen atoms are positively charged, thus the dye will be a cation with an

overall charge of +1, being in the red form.

• In the green form (pH of approx. 1) the dye will have no net overall charge (+2 and -2).

-

+Arg+

Precautions forChromophorogenic 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.

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Day 2, 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.

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Amino acids that absorb strongly in the UV.

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Garrett and Grisham, 3rd ed. Fig. 4.15

A typical protein: Lysozyme

2ZYP.pdb27

UV-absorbing amino acids

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6 Trp and 3 Tyr.

UV-absorbing amino acids

! 6 Trp and 3 Tyr.

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! 6 Trp and 3 Tyr.! Some are buried, others are stacked.

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 known1 :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 GLYASN TRP VAL CYS ALA ALA LYS PHE GLU SER ASN PHE ASNTHR GLN ALA THR ASN ARG ASN THR ASP GLY SER THR ASPTYR GLY ILE LEU GLN ILE ASN SER ARG TRP TRP CYS ASNASP GLY ARG THR PRO GLY SER ARG ASN LEU CYS ASN ILEPRO CYS SER ALA LEU LEU SER SER ASP ILE THR ALA SERVAL ASN CYS ALA LYS LYS ILE VAL SER ASP GLY ASN GLYMET ASN ALA TRP VAL ALA TRP ARG ASN ARG CYS LYS GLYTHR ASP VAL GLN ALA TRP ILE ARG GLY CYS ARG LEU

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! In our denaturing medium, at 280 nmεTrp = 5690 M-1cm-1 and εTyr = 1280 M-1cm-1.

J Biol Chem. 1963 Aug;238:2698-707

The experiment

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! 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 280 nm.

How we will do it

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Accuracy and precision(Day 1)

1. Use your pipettors to deliver 333 µl, 48µl, and 3µl onto a weighting paper, and report the weight of each drop.

2. Make up a series of standard sample dilutions.

3. Measure absorbance of each.4. Make a set of identical dilutions.5. Measure absorbance of each.

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What you have to do:

Step 1a. Use the pipettors in your locker and deionized water from the appropriate faucet near your desk (put some in a beaker). 1b. Go to one of the analytical balances, tare an empty boat then deliver 333 µl water. Record the weight. Repeat for 48 and 3 µl.

Accuracy (vs. concentration)

! Your T.A. will provide a concentrated stock solution of Bromophenol blue.

! Dilute with water to make the following series of derivative concentrations: 5%, 10%, 20%, 30%, 40%, 60%, 80% (all vol./vol.).

! Measure the absorbance at 590 nm of a cuvette containing only the water used as a diluant.

! Rinse the cuvette once with 5% solution and then measure the A590 of this solution.

! Repeat for the 10% solution, then the 20% ... etc to the highest concentration.

! Why should you read from lowest to highest concentration?

! Why should you rinse once with solution?

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Steps 2-3

Accuracy (vs. concentration)

! Plot A590 vs. concentration.! Identify the linear regime.! Use the known concentration of the

100% solution to calculate the extinction coefficient based on the linear portion of the plot.

! Use the absorbance measured for the 100% solution and the extinction coefficient to calculate the apparent concentration.

! How does your apparent concentration compare with the actual concentration?

! If you are making a direct determination of concentration and measure an absorbance above 1, what will you do to obtain an accurate value for concentration?

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Precision

! Make 5 ‘identical’ 20% solutions by making 5 independent dilutions of 100% solution. In all cases the final volume should be 3 ml.

! Measure A590 of each of your solutions and tabulate the results.

! Calculate the average value and the standard deviation for your set of readings.

! Comment on the results.

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Steps 4-5

Day 2, Experiment 1: Precautions for

Chromogenic 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.

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Day 2: juggling two activities.

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You will be doing two things today: a chromogenic assay and a direct determination.To use your time well you may want to interleave steps of the two activities. A flow chart helps to keep your mind organized as you hop back and forth.

Example:

Make series of standard samples

Add dye reagent. Start timing.

Make series of unknown samples

Add dye reagent. Start timing.

Read and record absorbances.

Read and record absorbances.

Measure A280 of lysozyme

Dilute lysozyme w. GuHCl

Measure A280 of lysozyme-GuHCl

Calculate concen-tration of diluted lysozyme-GuHCl

Calculate con-centration of initial lysozyme

Experiment 1

Experiment 2

Chromogenic assay

1. Make up standard sample dilutions as per table.

2. Make up unknown sample dilutions as per table.

3. Add 3 ml “Bradford reagent” to each. Start timer.

4. Gather cuvettes and get in line for spectrophotometer.

5. Once 5 minutes have elapsed read and tabulate A595. (Note how much time had elapsed.)

6. Do not leave without learning from your T.A. what the concentration of the standard is.

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What you have to do: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.)

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Chromogenic assay:How to do it

! The active ingredient is Coomassie blue G-250, 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:

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Vol. of Protein stdd. soln. (µl) Vol. of buffer (µl)

100 0.0

80 20

60 40

40 60

20 80

0 100

Making standard samples -1 Making standard samples -2! 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 into a small beaker and pipet from this aliquot. If there is left-over and it is not contaminated, pass it along to a colleague for use.

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! 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.

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Reading the standard samples

! Adapt the protocol detailed above to make a series of five samples of the unknown, plus a ‘null’ sample.

! Your notebook should include a sample table for the unknown that resembles the sample table you made for the standards.

! React the unknowns with diluted Bradford reagent as above and read them as above.

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Making the unknown samples

Tips for success• Be sure to mix all component

thoroughly before taking absorbance measurements

• The plots of absorbance vs. concentration in the second half of the experiment might not be linear. If so, identify the portion of the plot you will use as the basis for any analysis and explain why.

• If the color of your unknown tubes is more intense than the known tube with 100 µl of standard, tell the instructor so that the unknown can be remade.

Questions! 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 ?

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Day 2, Experiment 2:Direct determination of protein concentration

47

! We will determine the concentration of a lysozyme solution under denaturing conditions, 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 ε at 280 nm. Once ε is known the concentration of that protein can be determined without prior denaturation.

Direct determination of concentration.

1. Measure A280 of native lysozyme solution using a quartz cuvette.

2. Mix 2.5 ml of the lysozyme with 7.5 ml of guanidinium HCl

3. Rinse cuvette with 3 ml of lysozyme/GuHCl solution (discard solution used for this).

4. Measure A280 of 3 ml lysozyme/GuHCl solution.

5. Measure A280 of another 3 ml lysozyme/GuHCl solution.

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What you have to do:

Direct determination Protocol: how to do it.

! 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 cuvette 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 ? 49

(more detail on the steps listed above)

Questions to be answered in your theory section and

pre-lab.! 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.

! Why must we use a quartz cuvette ?

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Protocol, continued

! Transfer 3 ml of denatured lysozyme to the cuvette you just used and then discard this solution. (This step washes away residues of the previous sample.)

! Transfer a second 3 ml aliquot of denatured lysozyme to the cuvette and measure its A280 .

! Discard this solution too and transfer a third 3 ml aliquot of denatured lysozyme to the cuvette and measure its A280 (second measurement).

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Questions! How do your two readings for the A280 of

denatured lysozyme compare ?! How does it help to have two readings ?! What value should you use and how will

you know if it is valid ?

! Use your calculated extinction coefficient for denatured lysozyme to determine the concentration of denatured lysozyme.

! Now calculate the lysozyme concentration of the original native solution of lysozyme based on the dilution factor used.

! Use this and the A280 you measured for native lysozyme to calculate the extinction coefficient of native lysozyme.

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Post-lab thoughts! 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).

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