Biochemistry-Prep Of LDH Through Crude Tissue

Q. 1. Reasons for using too much of the beef heart tissue
The type of protein targeted in this isolation process (citrate
synthase) is usually found in larger quantities in the mitochondria of
cells with high metabolic rate compared to other tissues. Heart, being
one of the busiest organs is a high rate of metabolism especially within
the cells of its muscle. In addition, beef hearts are readily available
organs and the researcher can get them locally at a cheaper price. It is
necessary to use a large amount of heart tissue because there are many
types of proteins located in cells each of them in low concentration.
The large amount of tissue will help in isolation of a reasonable
quantity of the target protein.
The tissue is kept in cold to inhibit the metabolic action of enzymes
known as lysosomal enzymes to prevent them from destroying the sample
before the experiment.
The tissue is suspended in 0.2 M sucrose and pH 7.2 because sucrose
creates an isotonic medium, which prevents absorption of water by
organelles, thus protecting them from swelling, rupturing, and releasing
the content that is needed during the experiment. The pH 7.2 decreases
lysosomal activity, thus protecting the structure of the proteins
contained in the tissue.
When the tissue is homogenized, cells of the sample break open and
release cytosol and other organelles.
Q. 2. Subjecting the resulting beef homogenate to a series of
differential centrifugation steps help in sedimentation of organelles at
varying rates of centrifugation because they differ in size. The larger
cell debris and organelles sediment first, while the small ones sediment
by increasing the centrifugation speed.
Q. 3. Rationale for the use of two step ammonium sulfate addition
Different proteins have varying types of functional groups, which
results in differing solubility characteristics. This implies that the
solubility of protein differs with the concentration of salt. At a
concentration below the level required for precipitation of citrate
synthase, some proteins are salted out. Citrate synthase is then
precipitated and recovered by increasing the concentration of ammonium
sulfate and centrifugation.
Q. 4. Ammonium sulfate is avoided in dialysis buffer to prevent
osmolarity, which would affect the stability and conformation of
proteins. In addition, the removal of ammonium sulfate allows the
researcher to renature and solubilizes the proteins, which is
accomplished by dialyzing against a buffer solution that does not
contain ammonium sulfate. This allows the ammonium sulfate contained in
the sample to diffuse intro buffer solution until a balanced
concentration is reached. The process is repeated until the salt
concentration in the sample is completely reduced. Although both water
and buffer solution can be used to suspend proteins, suspending them in
water subjects them to higher chances of breakdown than when they are
suspended in buffer.
Q. 5. The instructions to collect the first fraction inform that the
molecules of citrate synthase are larger than the size of the
chromatography gel in use. In the size exclusion chromatographic
technique, small molecules enter the pores of matrix whole the larger
ones move around the matrix.
Uv absorbance at 280 nm is appropriate to monitor proteins contained in
elute fractions because proteins have aromatic side chains (mainly Tyr
and Trp residues), which absorb strongly at 280 nm.
Q. 6. The beads loaded on the cation-exchange column, which are
negatively, changed binds citrate synthase because it has positively
charged domains. Proteins and other debris that is negatively changed
pass through the column. Raising the pH of the mobile phase alters the
charge on citrate molecules, thus displacing them from the column.
Q. 7. There are several proteins having the same pI. These proteins
could have been focused on one band. In addition, SDS gel
electrophoresis functions by separating the different components on the
basis of their mass and this would result in the separation of any of
the polypeptides (as shown in the results) in the sample at the pI 5.6
band.
SDS gel electrophoresis should be done after isoelectric focusing
because SDS bonds tightly and uniformly along the polypeptide since it
is highly negatively charged. This makes it difficult to remove SDS from
protein, which reduces the integrity of the protein containing traces of
SDS. The protein loses its acid-base properties as well as native pI.
BIOCHEMISTRY-PREP OF LDH
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BIOCHEMISTRY-PREP OF LDH
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