Modern Experimental Biochemistry

Chemistry

Quiz 4 :

Isolation and Characterization of Bovine Milklactalbumin

Quiz 4 :

Isolation and Characterization of Bovine Milklactalbumin

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Each of the proteins listed below is treated with sodium dodecyl sulfate and separated by electrophoresis on a polyacrylamide slab gel. Draw pictures of the final results. (a) Myoglobin (b) Hemoglobin (two ? subunits, molecular weight = 15,500; two ? subunits, molecular weight = 16,000)
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In this problem, we are asked to draw a slab gel for four proteins:
1) Myoglobin
2) Hemoglobin B
3) Hemoglobin A
A slab gel will separate proteins based on their molecular weights - the heavier proteins will travel slower and thus less distance. A quick perusal of these proteins shows that myoglobin is the heaviest, followed by hemoglobin B and then hemoglobin A.
Thus, the slab gel will appear approximately as follows: img

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What methods of analysis could you perform on the purified ? -lactalbumin in order to determine the extent of purity?
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In this problem, we are asked to explain which methods of analysis are used in the experiment to determine the purity of the a-lactalbumin purified from the milk.
The process uses two chromatography steps (Sephadex [size exclusion] and affinity), and two different assays - SDS-PAGE and the Bradford Protein Assay. Chromatography is the method by which we purify, but it does not tell us the level of purity attained.
The Bradford assay is a subjective assay that tells us how much protein we have in a sample, but the SDS-PAGE separates the samples into their component proteins, which will tell us the the purity of our post-chromatography material.

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What amino acid residues in proteins bind to chromatography gels containing immobilized metal ions?
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In this problem, we are asked to state which amino acid residues bind the affinity column's immobilized metal ions.
The metal ion in the affinity column presents a positive cation for coordinating bonds with molecules that have electrons to donate. Conjugated ring systems are one such structure found in protein residues, specifically the imidazole ring in histidine and the indole ring in tryptophan. Sulfa groups such as the sulfahydryl group in cysteine also qualify.
Any residue with electron-donating groups (HIS, TRY, CYS, SER, and others) can be bound by the metal ions of the affinity column.

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img What physical characteristics of a biomolecule influence its rate of movement in an electrophoresis matrix?
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The dye ethidium bromide is often used to detect the presence of nucleic acids on electrophoresis supports. Explain how it functions as an indicator.
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What other purification techniques could be applied to the purification of ? -lactalbumin? Use a MEDLINE literature search. Study the following references for methods that have been applied to the isolation and purification of a-lactalbumin: W. G. Gordon and W. F. S'emmett, J. Amer. Chem. Soc. 75,328 (1953). W. G. Gordon and J. Ziegler, Biochem. Prep. 4, 16 (1955). U. Brodbeck, W. L. Denton, N. Tanahashi, and K. E. Ebner, J. Biol. Chem. 242, 1391 (1967). F. J. Castellino and R. L, Hill, J. Biol. Chem. 245, 417 (1970). L. Lindahl and H. Vogel, Anal. Biochem. 140,394 (1984).
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What would be the effect of each of the following changes on the relative mobility of ? -lactalbumin in SDS-PAGE? (a) Lower the pH of all buffers. (b) Increase the ionic strength of the buffers. (c) Change the temperature of the electrophoretic operation. (d) Increase the concentration of ? -lactalbumin. (e) Increase the concentration of bis-acrylamide in the gels. (f) Decrease the electrophoretic running time.
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img What is the main advantage of slab gels over column gels for PAGE?
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Why do most protein solutions absorb light of 280 nm wavelength?
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What assumptions must be made about the relative mobility of bro- mophenol blue when used as a tracking dye?
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Explain the purposes of protein and nucleic acid "blotting."
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Describe the toxic characteristics of acrylamide and outline precautions necessary for its use.
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Can polyacrylamide gels be used for the analysis of plasmid DNA with greater thaN3000 base pairs? Why or why not?
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the chemistry involved in the Bradford assay.
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Absorbance measurements of column fractions at 280 nm were used in this experiment to detect the presence of protein material. Do you think this method of analysis could lead to a quantitative determination of protein concentration? What other biomolecules might interfere with this measurement?
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Describe the procedure for obtaining a "difference spectrum" of isolated ? -lactalbumin vs. standard ? -lactalbumin.
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Comment on the validity of the following statement: "Ideally, the best Bradford calibration curve for this experiment would use purified ? -lactalbumin rather than gamma globulin."
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img Draw a slab gel to show die results of nondenaturing electrophoresis of the following mixture of proteins. The molecular weight is given for each. img
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img Explain the purpose of each of the chemical reagents that are used for PAGE. (a) acrylamide (b) N , N -methylene-bis-acrylamide (c) TEMED (d) sodium dodecyl sulfate (e) Coomassie Blue dye (f) bromophenol blue
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img Is it possible to use polyacrylamide as a matrix for electrophoresis of nucleic acids? What are the limitations, if any?
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