Biochemistry
9th Edition
ISBN: 9781319114671
Author: Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher: W. H. Freeman
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1. Know the structures of DNP-glycine and of the chromophore associated with cytochrome C.
Low Salt
Volume of blue band = 5.2 ml (Void Volume)
Volume of yellow band = 2.6ml
Volume of red band = 11ml
High Salt
Volume of blue band = 4.2 ml -> Void Volume
Volume of yellow band = 5.2
Volume of red band = 1.2
2. What is the exclusion limit and the fractionation range for CM-Sephadex resin used?
3. What are the ion exchange reactions which occur when the coloured mixture is first applied to the column under low salt conditions? What exchange reactions occur upon the change of buffer from low salt to high salt concentrations?
4. What were the values for Vo and Vi for this column? Where the values for Vo and Vi the same under both conditions?
5. Explain the order in which the various components of the mixture elute under each condition in terms of their physical properties and the nature of the CMSephadex G-50 column. Would the same order be observed with a G-50 Sephadex column (i.e. no CM component)?
6. Predict and explain the results that would have occurred if the same experiment had been run with a CM-Sephadex G-50 column equilibrated in and eluted with 0.1 M HCl or 1M HCl
7. Predict movement of protein hemoglobin: heme containing protein, 64,500 Daltons, reddish brown, pI=6.9 or vitamin B12: non-protein organic molecule, 1,355 Daltons, pink, pI=6-8 in the above two columns with blue dextrose, cytochrome c and DNP-glycine. If attempting this separation would you attempt to use more than two salt concentrations? Why? Would a different resin or cutoff be more appropriate.
Transcribed Image Text:1. Each group will be given a column.
Close the column and add a few mls of 0.1M potassium acetate buffer.
Using a Pasteur pipette, begin to add a well-mixed slurry of CM-Sephadex in
0.1M potassium acetate buffer to the column.
➤ Allow the columns to drip slowly while adding small amounts of the slurried CM-
Sephadex until the settled resin bed is about 2 inches from the top of the column.
Do not allow the column to run dry; i.e. do not allow the top fluid surface to
penetrate into the settled resin bed. Instead, add small aliquots of the acetate
buffer to the column to keep the fluid surface above the resin bed.
4.
2.
3.
Once the desired resin height has been obtained, stop the column flow when
about 1 ml of buffer remains over the top of the resin.
Immediately add 0.1 ml of the coloured solution (containing each of the
components in Table 1, see below) so that it forms a thin band on top of the resin.
Why does the solution sink below the buffer? (NOTE: Do not disturb the top of
the resin.)
Transcribed Image Text:5.
6.
7.
8.
> Start the column and keep topping up the buffer with 0.1M potassium
acetate buffer (pH 6.0).
Be careful not to disturb the top of the resin.
> Allow the column to continue flowing, replenishing the upper fluid
volume as necessary while observing the course of the elution.
Collect the eluent into a 10 ml graduated cylinder and note the
volumes at which each of the coloured solutions are first eluted from
the column. Make sure this data is recorded in your lab note book.
Once the second coloured reagent has eluted from the column replace the elution
fluid with the high-salt buffer (1.0 M potassium acetate, pH 6.0).
To change the elution fluid allow the buffer to run to the top of the resin and
immediately add a few drops of the high salt buffer. Allow this to run in and
repeat twice.
Then fill the column with the high salt buffer and continue the elution as
above in step 4.
Once the last component has fully eluted from the column, repeat the entire
experiment using the 1.0 M acetate buffer (what type of separation are you
know using)
To do this reapply the mixture from table 1 and elute using the high acetate
buffer from the beginning of the elution process (i.e. as described in step 4
above).
After the relative movements of all the coloured components have been observed
using acetate buffer, wash columns with 0.1M acetate pH 6, remove the columns
from their clamps, take them to a central container for used CM-Sephadex, and
empty the used gel-filtration ion exchanger into the container by shaking or
blowing out the resin. The resin will be regenerated by appropriate washings and
used again.
Complete Table 2 in your log book.
Table 1 Composition of mixture
Name
Blue dextran
Cytochrome C
DNP-glycine
Colour
blue
red
yellow
Molecular weight
>500,000
12,400
241
Ionic character
Strong anion at pH 6.0
but often reported as
non-ionic
pl = 10.7 net cation
below 10.7(strong
cation at pH 6)
pl=3.0 Anion above 3
(weak anion at pH 6)
2
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