lecture 8 - quantifying purity Flashcards
spectophotometry
- absorbance (A) = log (Io/I)
- Io = initial light passed through sample
- I = light transmitted and detected after passing through the sample
Beer-Lambert Law
- A = Ecl
- E = molar coefficient (L/mol/cm) - unique for each molecular amino acid - how much light can be absorbed at specific wavelength
- c= concentration (mol/L)
- l= path length (usually 1cm)
- Trp > Tyr > Phe - only ones that absorb UV
B-L is useful for determining the concentrations of what proteins?
pure proteins only
B-L is not useful for:
determining concentrations in a mixture of proteins
B-L: for a crude mixture of proteins with a typical or average amino acid concentration, absorbance is equal to:
-A280 = [total protein] in mg/mL
-[P] = 1 mg/mL then A280 = 1
(just approximate and only for mixtures of many proteins)
what is a method for measuring total protein?
Bradford assay
how does a bradford assay work? is it dependent on amino acid composition?
- uses coomassie die which binds protein and absorbs 600nm
- more sensitive
- not dependent on amino acid composition
- use a standard curve to measure total protein (look at absorbance on y axis then follow to mg/mL on x axis)
what assays are useful for the specific measurement of target protein?
- immunoassay
- enzyme assay
what does an immunoassay detect?
antibodies that bind to the target protein
what does an enzyme assay measure?
the reaction catalyzed by target protein
- if Sāā>P by a target enzyme
- then can measure increase [S] or [P] using spectophotometry
what is an enzyme unit?
1 unit = amount of enzyme that catalyzes the conversion of 1umol of S in 1 min at 25 degrees C
specific activity equation
units/ total protein in mg
what happens to specific enzyme activity as purity increases?
specific activity of a protein increases up to a maximum point where the protein is pure
what does # units measure? (purity or yeild)
yeild
what does specific activity measure? (purity or yeild)
purity
example question:
what is the specific activity of an extract with 250 ug protein that converts 1500 umol of S in 30 minutes
units = 1500 umol/30 mins = 50 umol/min
200 units/mg
s.a. = 50 units/250 ug = 50 units/0.25 mg = 200 units/mg
how do we analyze a purification table to determine the largest increase in purity?
compare the difference between specific activity for each step, the largest difference provides the step that caused the largest increase in purity
how do we analyze a purification table to determine the largest loss (lowest yeild) of protein?
compare the differnce between enzyme activity units - the latgest difference provides the step that caused the largest loss
how do we analyze a purification table to determine which purification step is unneccesary?
compare the specific activity, which ever step produced the smallest increase is unneseccary
