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Week 6 Homework – Calculation of Mass Extinction Coefficient of L7AE
Assignment
Compute the mass extinction coefficient for L7Ae. Include these calculations in your
notebook next week (week 6).
Why are we doing this calculation?
In a few weeks, we will be using spectroscopy (via the Nanodrop apparatus) to
determine the amount of protein we have isolated. To do this accurately, we need to
compute the mass extinction coefficient of the protein we have designed. The
Nanodrop uses a general mass extinction coefficient (of 1) if you do not enter one in.
With the Nanodrop, we can measure the absorbance of light that a sample has at a
particular wavelength of light. The mass extinction coefficient tells us for an
increase in absorbance of a material, how much the mass has increased. The mass
extinction coefficient of a material is how strongly the substance absorbs light
at a given wavelength per unit mass (in a constant volume).
How to compute the mass extinction coefficient
The mass extinction coefficient for a protein is equal to the molar extinction
coefficient divided by the molecular weight of the protein.
Molar extinction coefficient
 - molar extinction coefficient (Abs*mol*Liter-1*cm-1)
A - Absorbance of the protein at wavelength  (Abs)
L – light path length (cm) – in this case, for the Nanodrop, L =1 cm
M – molar concentration of absorbing material (mol*Liter-1)
A
 
ML
Total absorbance of a molecule
The total absorbance of a molecule can be calculated as the sum of the absorbances
of its individual components.
AT  Total absorbance
Ai  Absorbance of component i
si  Number of component i



i
AT   si Ai  s1 A1  s2 A2 K  si Ai
1
Mass extinction coefficient

X = molecular weight of protein (g/mol)
Mass extinction coefficient =

x

Tools to help
With the ProtParam tool, you can find out 
the molecular weight of a protein and it’s
amino acid composition. Note that ProtParam makes the assumption that all
cysteines participate in a cystine, a disulfide bridge formed by the oxidation of two
cysteine residues. We need to correct for this ProtParam assumption.
Why do we need to correct ProtParam’s calculation of the extinction coefficient?
Notice that only disulfide bridge (cystine) absorbs at 280nm, cysteine DOES NOT
absorb at 280nm. We presume that inside the cell, the conditions are fully
reducting, so we assume that there will be NO disulfide bridges (cystines) in our
protein.
ProtParam tool:
http://ca.expasy.org/tools/protparam.html
Protparam documentation:
http://ca.expasy.org/tools/protparam-doc.html
Useful parameters
Only a few amino acids have significant absorbance at a wavelength of 280nm.
280 TRP = 5502
280 TYR = 1490
280 disulfide bridge (Cystine) = 125
Protein sequence
Below is the sequence of the protein that has been expressed in the lab. Use this
entire sequence in your calculations. The colors are simply for your reference to
know the different parts of this protein that we have engineered.
The his tag will be used during purification; histidines will bind to the cobalt in the
affinity columns and other proteins will flow through the column and be discarded.
To release the protein, imidazole is used as a competive binder to the cobalt. SUMO
is recognized by SUMO protease. SUMO protease recognizes SUMO and will cut the
recombinant protein, leaving the native L7Ae (protein as it would be found
naturally).
Red – His tag
Blue – SUMO protein
Green – Pyrobaculum oguniense L7Ae protein
Black – sequence
MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFK
IKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG
GMAVTIDPKTFYANPLPGKPFYVRFEVPSDVAEKALEILSIAKQTGKIKKGTNEATKAV
ERGLAKLVLIAEDVDPPEVVAHLPLLCEEKKVPYVYVPSKEKLGKAAGINVSAAAAVVI
EPGQAAGELEALVSKINEIRAKNGLNAIPLPAGARR