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Final

BCH3346 Final: BCH3346 Final Exam Study Guide


Department
Biochemistry
Course Code
BCH 3346
Professor
Jos�e Coutu
Study Guide
Final

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Experiment 0: Protein Quantitation
Learning objectives
1) Explain why the bradford Assay must be preformed under acidic condition
2) Compare and contrast three forms of CBB-G250 that can exist
Protein Quantification
The bradford assay is a dye-based spectrophotometric assay which allows for the
determination of protein concentration in an aqueous sample. Essentially, this means
that a colour change is produced when protein-dye complex forms, this change in colour
of the dye can be detected as a shift in wavelength, measured by a spectrophotometer.
Free BBG dye exists in three forms:
1) Cationic form that absorbs at 470 nm (red) - ACIDIC
2) Netural form with an absorption maximum at 650 nm (green) - NEUTRAL
3) Anionic form that absorbs at 595 nm (blue) - BASIC
Because it is the anionic form (blue) that non-covalently binds to protein, the equilibrium
shifts from the cationic form (red) to the anionic from (blue) as BBG binds to proteins.
Thus, the cationic form acts as a reservoir of the binding dye. The reaction is preformed
under acidic conditions (pH < 1) so that BBG is mostly in the cationic, form that absorbs at
470 nm (red). The two other forms are also present, although in lower proportion.
The binding can take place at neutral and alkaline conditions, but at these pH the anionic
form (blue) is much higher and its contribution to the absorbance at 595 (background
noise) is too high for a sensitive assay.
Note this effectively answers questions 1&2
Protein Standards
In order to determine the total protein concentration in an unknown sample, one must
compare its absorbance to that of a known concentration of protein. This is easily
achieved by creating a standard curve of absorbance (A595) Vs. Concentration using a
known amount of a protein standard, in our case the protein standard will be Bovine
Serum Albumin (BSA).
When preparing the standard curve, choose concentrations of BSA ranging from 0 to
100mg/mL. The absorbance at 595nm of the standard are then recorded and a A595 Vs.
Concentration of BSA (µg/mL) standard curve is produced. Because absorbance varies
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linearly with concentration (recall Beer-Lambert law: A =ecl) and e and l are constant, we
can draw a line of best fit and produce the linear regression equation : y=mx+b, where x
represents the concentration in µg/mL.
Finally, determine the absorbance of the unknown protein solution dilutions and plug in the
value for y to determine the concentration in ug/mL, x.
Dilution Factors
Important! When stating the final concentration, or even when plotting the concentration
of the standard curve, you must ensure you correct for any dilutions by multiplying through
by the dilution factor.
Experiment 1: Enzymatic Characterization of Heart Transaminases
Learning objectives
1) Explain the role of transaminases in amino acid metabolism
2) Differentiate between enzyme activity and specific activity
3) Explain the underlying principles of coupled assays
4) Discuss the premise of the equilibrium constant and enzymatic assay
Transaminases
Transaminases catalyze the transfer of an amino group (-NH2) from one amino acid to a
keto acid (such as pyruvate and oxaloacetate). This reaction is notable because it occurs
without yielding free NH3 - the amino group is retained through transfer to vitamin B6 (a
prosthetic group within the transaminase). Also note that the transamination reaction is
reversible (Go ~ 0).
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The specificity of the reaction is referred to by the naming of the enzyme:
Glutamate-Pyruvate-Tranaminase (GPT):
-Catalyzes transfer of amino group from glutamate to pyruvate
-Produces alpha-ketoglutarate and alanine
Glutamate-Oxaloacetate-Tranaminase (GOT):
-Catalyzes transfer of amino group from glutamate to oxaloacetate
-Produces alpha-ketoglutarate and aspartate
Role in Metabolism
Note this effectively answers questions 1
Transaminases play a very important role in amino acid metabolism
Amino Acid Catabolism & Elimination of Nitrogen Via Urea Cycle
Amino acids transfer their amino group to α-ketoglutarate to form glutamate (i). Then the
glutamate is either converted by the action of GOT into aspartate, a precursor of the urea
cycle (ii) or into NH4+ by action of GDH (iii). Urea excretion is the pathway used for
elimination of nitrogen by most terrestrial vertebrates.
Amino Acid Catabolism & Glucose Synthesis
GPT is responsible for the conversion of pyruvate into alanine (iv) which is delivered to the
liver where it is used for glucose synthesis.
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