BEYOND THE BASICS:
FACILITATION OF ADVANCED TOPICS IN BIOCHEMISTRY
NSFILI-IP Award 1997 - 1999
DUE 9750698
Final Report Filed 2/5/2000
Sandra L. Jewett, PI
Advanced Topics
Demonstrated
| LABORATORY EXERCISE | NEW PRINCIPLES DEMONSTRATED | IMPROVEMENTS |
|
Isolation of b-carotene (Chem 464, 462, 365) |
Spectra of beta-carotene vs vitamin E | Students collect and compare A vs data for important dietary compounds |
|
Chymotrypsin catalysis (Chem 464, 462) |
pKa of histidine determined from rate data | Students determine most likely functional group that is involved in enzyme catalysis |
|
Chymotrypsin catalysis (Chem 461) |
Spectra of p-nitrophenol
product vs pH (pKa of p-nitrophenol product) |
Students learn that product spectrum is pH dependent, and why a pH correction is required for data, and determine pKa of product |
|
Wheat Bran Phosphatase (Chem 365) |
Spectra of Protein
Extracts A vs for typical proteins; A vs for tryptophan, tyrosine, and phenylalanine |
Students learn which amino acids determine protein absorption in the UV |
|
Lactate Dehydrogenase (Chem 464, 461) |
Spectra of Protein
Extracts A vs for typical proteins; A vs for tryptophan, tyrosine, and phenylalanine; compare to bovine CuZn superoxide dismutase which has no tryptophan |
Students learn which amino acids determine protein absorption in the uv; compare spectrum of typical protein to unusual protein |
|
Lactate Dehydrogenase (Chem 461) |
1/vo vs 1/[S1] at three [S2]; |
Students learn kinetics of two substrate reaction |
|
H+ Pumping by Leaky Chloroplasts (Chem 462) |
NEW: Spectrum of chloroplast suspension in 80% acetone. |
|
|
Directed Undergraduate Research (Chem 495) |
Directed Undergraduate
and MS Research Modern methods of data collection and analysis |
Software compatibility, reliability, expanded future possibilities.
Lowry and Bradford Protein Assays The Infamous Hyperbolic Equation in Biochemistry |
Spectrum of beta-carotene versus vitamin E
Students isolate and quantify
b-carotene (or mixture of carotenoids) from carrots. By
comparing the spectrum of their extracts to authentic b-carotene
and vitamin E, students learn that have characteristic spectra
and that these spectra can be used to help identify biologically
important compounds.
(Chem 464, 462, 365)
pKa for active site histidine for chymotrypsin catalysis of hydrolysis of p-nitrophenylacetate
Students determine the pKa
of an active site functional group from the rate of the
enzyme catalyzed hydrolysis of the ester substrate as a
function of pH. Using these data, students determine which
amino acid side chain is most likely the functional group
involved in enzyme catalysis.
(Chem 464, 462)
Spectrum of p-nitrophenol as a function of pH to determine the pKa of p-nitrophenol
Students take spectra of samples
of p-nitrophenol as a function of pH. Students learn
that the spectrum of the product of the chymotrypsin catalyzed
hydrolysis of p-nitrophenylacetate is pH dependent. This
reinforces an understanding as to why a pH correction is required
for their rate data as well as for determination of the number
of active sites of chymotrypsin. Students also determine
pKa of this product, a value used in the correction term (1 +
[H+]/Ka).
(Chem 461)
Students examine a spectrum of
the wheat bran phosphatase extract which demonstrates a typical
spectrum of most proteins. They compare this spectrum to
spectra of tryptophan, tyrosine, and phenylalanine to learn which
amino acids determine protein absorption in the UV.
(Chem 365)
Students examine a spectrum of
the rabbit muscle extract which demonstrates a typical spectrum
of most proteins. They compare this spectrum to spectra
of tryptophan, tyrosine, and phenylalanine and to bovine CuZn
superoxide dismutase which has no tryptophan. Students learn
which amino acids determine protein absorption in the UV.
(Chem 464, 461)
Students carry out classical Michaelis-Menten
enzyme kinetic analyses with rabbit muscle lactate dehydrogenase.
From their data of 1/Vo vs 1/[pyruvate] at two or three
[NADH], students learn how to determine the order of binding of
these two substrates.
(Chem 461)
NEW: Spectrum of Chloroplast Suspension
Students determine the amount of chlorophyll in a chloroplast suspension after extracting a small portion of the chloroplasts with an 80% acetone solution and determining the absorbance at 652 nm. A spectrum of this solution is also obtained and compared to the spectrum of a carrot extract (obtained previously). The b-carotene is seen to be present in the chlorophyll extract.
Directed Undergraduate and Master of Science Research
Students carry our directed research projects and learn modern methods of data collection and analysis with software that is compatible with EXCEL. They learn about data reproducibility as well as experimental design. (Chem 495)
The Lowry Protein
Assay Revisited (PDF)
The Bradford
Protein Assay Revisited (PDF)
The
Infamous Hyperbolic Equation in Biochemistry (PDF)