6. (10 points) You have isolated a protein and are in the process ...

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6. (10 points) You have isolated a protein and are in the process of characterizing it. You have obtained the data presented below. Analysis on a gel chromatography column indicates that it has a rather high filtration molecular weight of about~150kDa. When you run this protein on an SDS-PAGE (without 2-mercaptoethanol or other reducing results. agent), it runs as a single band with a molecular weight consistent with the gel filtration When you repeat the SDS-PAGE in the presence of 2-mercaptoethanol, you now find that the once large protein runs as two bands. Use the following data to determine their molecular weights. A blank graph is provided below if needed. If you do use it, apply appropriate labels. a) What can you deduce about the quaternary structure of this protein? (type and number of subunits, organization, arrangement, etc. - make a sketch) b) Can you make any conclusion about the interactions between these subunits? 11. (5 points) For a Michaelis-Menten reaction, the individual rate constants are: k1 =7 x 107 M-¹sec¹¹, k2 = 2 x 104 sec¹¹ and, k-1 = 1 x 10³ sec¹¹. What are the values of KM and Ks (note, Ks is the Kd for substrate binding)? In this catalyzed reaction, does substrate binding approach equilibrium or does the system enzyme behave more like a steady-state process? Why? 4. (20 points) Shown below is the amino acid composition of histone H4, the smallest of the histones isolated from calf thymus and one of the most highly conserved proteins known. It is essential for DNA packaging in the eukaryotic nucleus. Number of Mass Amino Acid Residue Residues Residue Weight Contribution Alanine 7 71.0 497.0 Arginine 14 157.2 2200.8 Asparagine 2 114.1 228.2 AsparticAcid 3 114.0 342.0 Cysteine 103.1 0.0 GlutamicAcid 4 128.1 512.4 Glutamine 2 128.1 256.2 Glycine 17 57.0 969.0 Histidine 2 137.0 274.0 Isoleucine 6 113.1 678.6 Leucine 8 113.1 904.8 Lysine 11 129.1 1420.1 Methionine 1 131.1 131.1 Phenylalanine 2 147.1 294.2 Proline 1 97.1 97.1 Serine 2 87.0 174.0 Threonine 7 101.1 707.7 Tryptophan 186.2 0.0 Tyrosine 4 163.1 652.4 Valine 9 99.1 891.9 HOH 18 Total 102 Mol.Wgt.= 11249.5 i) If you were trying to generate a series of peptides from this protein for sequencing, which of previous enzymes would be most useful? Consider this carefully and justify your choice?

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Problem 6.

Answer (a &b):
Gel filtration and SDS-PAGE (under non reducing conditions) - both these techniques are used to quantify the relative molecular size of the native protein. Molecular sizes are calculated by comparing their movement to the marker proteins- that’s why ‘relative’. These techniques do not reveal the sub unit composition and absolute MW. Absolute MW can be calculated once the protein sequence is known.

SDS-PAGE under reducing conditions results in the reduction of all the disulphide bonds between the subunits of a multimeric protein, so we can calculate how many different kinds of ploy peptides are present in the multimeric protein. Combining the results from both the techniques, we can calculate how many different polypeptides are there in a multimeric protein and how many copies of each poly peptide are present.

Coming to the present problem,
MW calculated by gel filtration= Single peak corresponding to ¬150 KDa
MW calculated by SDS PAGE (Non reducing) = Single band corresponding to ¬150 KDa
Number of bands in SDS PAGE (Reducing) = 2 (assume A & B)...

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