BabcockHall

Stay away from E-64 and leupeptin. Look into regenerating cysteine protease activity after treatment with PMSF. I think dithiothreitol will work, but I don't have a citation.

What's wrong with the MAP kinase cascade? I am not sure why you are posting this question in homework help. Is this homework or research?

The explanation may have some truth in it, but I have to wonder whether or not the whole story is exactly what they imply. The interactions among the bases in ds DNA is not classically hydrophobic in nature, if I recall what I read in Voet and Voet correctly. Of course, the interactions between aromatic groups on the membrane with the bases of DNA or RNA might still be hydrophobic, but this should ideally be demonstrated.

I once lyophilized protein that had been dialyzed against 1 mM hydrochloric acid. I followed the lyophilization with complete hydrolysis of the protein. Perhaps 1 mM hydrochloric is not suitable for your protein, in which case ammonium formate sounds reasonable.

It does not seem to me that you have provided enough information to answer the problem.

Do we know whether this protein has a single alpha helix as the transmembrane domain, or are there multiple alpha helices? A few proteins have beta transmembrane domains also, but perhaps we can ignore them. I think your answer might be backwards.

I am not certain. I think that citrate would also activate acetyl CoA carboxylase, which would promote fatty acid biosynthesis. That is little more than speculation on my part, however.

I have never heard of demand reactions. Flux is sometimes used in metabolic control analysis, and for a pathway operating under steady state conditions, the overall flux through the pathway is equal to the rate of each step. Rates are typically given in units of concentration over time. I could also see flux being used as you suggest, but I have not encountered it myself.

In general rate constants can be found in two ways: by calculating the initial rate or by studying the integrated rate equation. For a crude estimate of k, I would try the former approach, but it is going to be very rough. Too much of A has been consumed for this approach to be perfectly valid.

@OP 7. and 8. Do you know the specificities of trypsin or chymotrypsin? What are the chemical properties that each protease is looking for? 5. Do you know which amino acid residues have side chains that are charged? Do you know the charge on a cation exchange resin? An anion exchange resin?

The covalent changes I had in mind were not limited to proteolysis. I was also thinking of oxidation of a methionine residue to a methionine sulfoxide, for example. Asparagine residues can undergo certain changes as well, but I don't have a citation handy. These covalent changes are somewhat different from noncovalent changes brought about by heating or other means. Some (but not all) unfolding events are reversible; many of the covalent changes are not reversible.

LD, Thanks. I should have said that my question was directed to the OP to help with question 4. Penicillin reacts covalently with the enzyme in question (sometimes called transpeptidase), which is not true of all competitive inhibitors. Sometimes people include one or more additional classes when discussing inhibitors of enzymes. One such class is irreversible inhibitors, and both penicillin and asprin fall into this category.

With respect to 4, what do competitive inhibitors generally do?

I have never thought about it in those terms, but I suppose you are right.

It could be that the question is related to the Bohr effect.

I would not say that the membrane has a hole poked into it, exactly. As long as both the conjugate acid and the conjugate base forms of the uncoupler are soluble in the membrane, they should transport protons down their electrochemical gradient.

The problem specified that nitric acid and sulfuric acid were in excess (sulfuric was probably a catalyst, anyway). Therefore they cannot be limiting reagents by the assumptions of the problem.

Noncompetitive inhibition is sometimes seen when an enzyme binds two substrates and the inhibitor resembles the substrate that is not varied. For example adenosine diphosphoribose (which resembles a portion of NAD) is a noncompetitive inhibitor versus formate with respect to the enzyme formate dehydrogenase. Some books refer to noncompetitive inhibition as mixed inhibition, potentially leading to confusion. Noncompetitive inhibitors bind in the presence or the absence of substrates.

Yes, hydroxide ion binds very weakly to Dowex 1 (quaternary ammonium ion), more weakly than formate and much more weakly than chloride ion. Many manufacturers give suggestions for how many column volumes of water to use and how to test for completeness. One such guideline is to use four column volumes of water and see that the pH has returned to less than 9. Ethanol is not an ion and should not displace hydroxide ion.

Truthfully, I don't understand the question. Suppose you are cleaning a Dowex 50 column with NaOH. The Dowex will now be in the Na+ form. If you add a buffer with a different counterion, your exchanger will be in a different form. Even if you make sure you don't run afoul of this problem, you will still need water to rinse out the buffer. Why would water rinse out buffer salts any more efficiently than it does NaOH?

Making an ester from an acid and an alcohol (such as methanol) might be fast or slow under your conditions. However, acetone is not an alcohol; therefore, I don't see how it could form an ester with formic acid. Perhaps I am missing something.

I don't know the exact number. Some oxaloacetate is simply a catalyst, as are all of the tricarboxylic and dicarboxylic acids of the TCA cycle (citrate, succinate, etc.). However, the way in which excess oxaloacetate could be consumed by the TCA cycle involves first converting it to phosphoenolpyruvate (PEP). Why don't you attempt a calculation based on that?

What is the specificity of the enzyme from bacteria, as opposed to ribonuclease A, for example? Which assays have you considered so far?

Robyt and White's book (Biochemical Techniques, pp. 151-154) states that gels range from 0.6% for DNA having 1-20 kilobases t0 2% 0.1-0.3 kilobases. They also give similar suggestions for sequencing gels; in other words, the percentage of agarose depends upon the size of DNA molecule being separated.

In protein separation the best percentage of acrylamide depends on the molecular weight of the peptides being separated.