Last week all the excitement was about activity. The radishes were homogenized, and we measured the activity. The crude homogenate was fractionated with ammonium sulfate, and we measured the activity.

Activity, activity, activity. All about the activity. And for good reason as that activity is the one, sole handle or measure we have on peroxidase. If we hadn't measured activity, you probably wouldn't have believed me that there was any peroxidase at all.

But peroxidase isn't the whole story. Sure, it is the principal, the lead, our Hamlet. But remember, Rosencrantz and Guildenstern were in the production too (and lived!).

So, who are our Rosencrantz and Guildenstern—proteins! We are working to purify peroxidase from the other proteins in the radish. We have been working to remove all proteins except peroxidase.

So, how do we know if we've done that?

There's an assay for that.

Today we'll be using the Lowry assay to determine total protein concentration. The basic principle of the assay is that copper ions in alakline conditions react with peptide bonds. The peptide bond–copper ion complexes are reduced with Folin-Ciocalteu reagent to develop a blue color in proportion to the amount of protein in solution.

Relatively simple, but how is it calibrated?

A standard curve.

That means we'll need a purified stunt–protein for the assay for which we already know the concentration. That will allow us to link protein concentration to blue color for the standards. And allow us to calculate a real, honest–to–goodness protein concentration from the blue color of the assay run on our samples.

Easy peasy lemon squeezy (remembering, of course, that we squeezed lemons earlier this semester).

Not so fast. Isn't there going to be a catch? There is always a catch, right?

Well, yes, there is one little wrinkle.

(NH4)2SO4.

As it turns out, ammonium sulfate does interfere with the assay at very high concentrations. Not a hard problem to overcome, but one that we must be mindful of as we setup the reactions. For every solution in more than sixty percent [(NH4)2SO4], we'll need to dilute (remember dilutions?) 1:1 with phosphate buffer. That'll reduce the [(NH4)2SO4] to tolerable levels and only require a minor dilution calculation (remember those?) to account for during analysis.

0. If and only if you have samples in more that 60 % [(NH4)2SO4], remove 500 μL of that sample to a microcentrifuge tube and dilute with 500 μL of phosphate buffer. This will be the sample you use for protein determination below since it is now low enough in ammonium sulfate to not interfere with the reaction. Do be sure to note which samples you have diluted as you will need to account for this dilution during data analysis.
1. In five 13x100 mm glass tubes, add 0.00, 0.05, 0.1, 0.2 and 0.3 mg of bovine serum albumin. Note that the stock solution is at a concentration of 1 mg/mL so that you can easily measure these masses by volume. For each sample, bring the total volume to 0.3 mL with phosphate buffer. The tube without protein will serve as your blank for the spectrophotometer.
2. In four other tubes, place 0.1 mL samples of your crude homogenate, AS-I (first pellet), ammonium sulfate cut and AS-III (what was never made into a pellet). Bring each of these tubes to 0.3 mL with phosphate buffer.
3. Add 6 mL of alkaline copper reagent to each of the 9 tubes, mix and let stand for 10 minutes.
4. Add 0.3 mL Folin-Ciocalteu reagent to each of the 9 tubes, mix and let stand for 30 minutes. It is safe to leave lab during this break, your tubes will not run away.
5. After 30 minutes, blank your spectrophotometer with the protein–less sample (and don't forget that the zero [protein]–zero absorbance point will be on your standard curve).
6. In turn, measure the absorbance of each sample.

Prepare a standard curve of absorbance as a function of protein concentration (don't forget to include the zero–zero point). Fit a linear function to your points and extract the equation for your standard curve. This will be a figure in your paper, don't forget to write a good figure legend detailing what you've done in making the standard curve.

Using your standard curve, determine the protein concentration in each of your samples. Be sure to account for the dilution in those samples in which you made a dilution for the ammonium sulfate. For all samples, remember that you diluted 0.1 mL to 0.3 mL in preparing the assay. Be sure to account for this dilution in all samples.

For your data (class data analysis to come), put it together into a table like the one below.

Table 1. Typical prototype of a purification table with calculation hints.

Sample	Volume (mL)	[Protein] (mg/mL)	Activity (U/mL)	Total Activity (U)	Specific Activity (U/mg)	Percent Yield (%)	Fold Purification (X)
Crude Homogenate	#	#	#	*	§	100	1
Ammonium Sulfate Cut	#	#	#	*	§	†	€

Complete the table according to the following calculation rules:

• #   These are measured values that you can fill in from your data from week 1 or week 2. Do note that last week you calculated U. Those units were in 0.020 mL of sample used in the assay. Division will give you U/mL.
• *   The total activity is activity (U/mL) multiplied by the total volume (mL).
• §   Specfic activity is a measure of the amount of activity relative to the amount of protein. Through a purification, if you keep your enzyme and remove other protein, the specific activity should go up. You'll know your done when the specific activity doesn't go up anymore—since all the protein is your enzyme. Do note that for most of you, this number will not look so good (not higher) for your cut. Don't worry, there will be, at most, one group who tried the ideal concentration of ammonium sulfate. There could also be no groups that win. The specific activity (U/mg) are calculated by dividing the activity (U/mL) by the protein concentration (mg/mL). The volume units in both the numerator and denominator cancel, and you are left with U/mg.
• †   The percent yield is the total activity of the sample divided by the total activity of the crude time 100 percent. The yield is based on following your enzyme, which is measured by activity. Remember many of you will not have peroxidase in your cut, do not be alarmed.
• €   Fold purification is based on the increase of specific activity in each step. That is specific activity of this line divided by the specific activity of the crude. In a purification, specific activity climes and plataeus, but remember that you may not have enzyme in your cut.

The above table should be in your results section.

The last bit of data that should be in the results section is the class aggregate data. The question is what concentrations of ammonium sulfate would you want to use as a step in the peroxidase purification?

The key thing to look for is which cut kept the most activity. You may include the table below directly, or consider turning it into a figure of your own design. You can use the raw activity numbers or calculate something like a percent yield or fraction retained for each cut. Define your own measure, you're allowed.

Table 2. Student groups, ammonium sulfate percent cut and activity measurements of crude and ammonium sulfate percent cut.

Lab Group	(NH4)2SO4 Cut (%)	Crude Activity (U)	(NH4)2SO4 Cut Activity (U)
Kirk & Poole	20–25	0.1749±0.0016	0.0933±0.0007
Anderson & Brown	30–35	28400±5000	21200±3500
Miller & Springer	40–45	8571.42±3544.36	4586.46±344.56
Bianchin & Buehler	50–55	17210.53±3947.37	744.36±473.68
Hershberger & Terhune	60–65	35413±12660	4735±1884
Chen & Leak	70–75	–	–
Cramer & Hale	80–85	–	–
Baksa & Farcas	90–95	27293.2±6152.56	9323.23±1406.65

Ok, so we have all our data and we're ready to write.

Every paper has a title which attempts to cover the system (peroxidase), the basic question (purification) and the major conclusion (we might learn something about purification). You can be positive or negative here. It is often far easier to show how something doesn't work than to show how it does. Since we're only writing part of a paper, we may not have all the answers. Be honest.

The abstract is 250 words of complete summary (see, there is a reason you've been working to that length all semester). It should cover the question, the system, a bit of the methods, results and conclusions about what it all means. There is a bit of art getting all of that into what is a modestly sized paragraph, but since you've been working toward that target all semester, you should at least know the challenges.

While the title and the abstract appear first in the paper, they should probably be the last things you write as they cover the full contents of the paper.

The best way to describe an introduction is to think back to how you were taught to write an introduction when you were 10 or 12 or 14 or 16 (no shame). The introduction is there to bring the reader from the very big question to the particular problem you wish to answer in the paper.

There are a couple places that I might start the introduction. One of those is protein purfication. As Arthur Kornberg was quoted last week, biochemistry "is only by purifying enzymes." Thinking about purification you might address methods, necessity or why you might enjoy having a purified protein.

You might start with the power of plants. Nature's solar panels. Almost everything you eat originates with plants (maybe mushrooms and Marmite are exceptions). Understanding plants will greatly help feed the world.

You could start with peroxidase activity. Peroxidase functions, in part, to eliminate reactive oxygen species. Molecules with which you also must deal. There is a potential medical start; study peroxidase to understand human disease.

Or you could start with the research aspect. Peroxidase, since it makes a variety of colorful redox products, is often used as an enzyme marker for some other event. Peroxidase is used as a reporter gene to identify gene expression. Peroxidase is coupled to antibodies to identify binding. There is also the pure research aspect which might be your first sentence and theme.

Here is a review paper (like short text book chapters, not papers that present new experiments) papers which will detail peroxidases (there are many) to help with the introduction. If you cite this (or any other) paper, use the style of the journal Biochemistry for your references.

The methods section describes everything you have done in enough detail that someone familiar with laboratory could reproduce your work. This section is divided into subsections, one for each method. In our case that would be homogenization, ammonium sulfate precipitation, the measurement of activity and the measurement of protein concentration. You can be brief here since all of the methods are above. One paragraph for each will do.

This section has the data, largely in figures and tables with enough prose to connect it all together.

If you'd like to make some figures and tables early, you can certainly work with the sequence and structure of peroxidase. The sequence of our most likely peroxidase is here in FASTA format. Some thing you might want to answer is what is the molecular weight of peroxidase. Small proteins are about 12,000 g/mol, large complexes are 500,00 g/mol or more.

You might also be interested in calculating the isoelectric point such that you could suggest alternate purification methods based on column chromatography. What ion exchange resin might you use at which solution pH?

You might also think about similar sequences and what makes a peroxidase a peroxidase. Using BLAST you should be able to find other peroxidase sequences. Collecting several of those you could demonstrate the key elements of peroxidase with a multiple sequence alignment.

A structure of peroxidase from radish is in the Protein Data Bank as 4A5G. You might wish to include a ribbon diagram as a figure to illustrate the protein. The centeral activity is around the associated heme group (a central iron atom, not surprising for redox chemistry, and a modest organic structure). Illustrating the heme group along with the secondary and tertiary structure would go a long way to presenting a complete view of the enzyme.

These data should absolutely, positively and without a doubt be in your paper:

• Standard curve for [protein] determination from this week.
• The completed version of Table 1 above. Include the calculation which get you here: units of activity, standard curve equation and calculations explicitly for completing Table 1.
• Table 2 (class data) or some data presentation derived from the data in Table2. If you are plotting points with errors, the classic presentation is to plot the mean as point with a vertical bar up and down scaled to the size of the error (standard deviation). (Hint: in Excel, you'll want your errors in the table with your data and use the "Custom" "Error Amount" to point Excel to your standard deviations.)

In the discussion, you will revisit each item of data and, in light of the topics in your introduction, explain, interpret and extend what those data mean. Since our major question is which [(NH4)2SO4] range makes the best "cut," you'll want to think about finding maximal activity in that fraction as a function of [(NH4)2SO4] (and you might even want to make that plot—yes, you can introduce figures which synthesize results in the discussion).

You'll also want to account for how the protein is fractioned (recall you have more protein concentration data than you have used in the purification table). Relative to the first pellet and how much protein remains in solution, how much protein did you get in your cut? Ideal situations precipitate very little protein in the cut with large amounts in the first pellet and still in solution. It is perfectly fine to introduce a figure around this area if it is summary and helps you make an argument.

The discussion can also include speculation. Maybe you've noticed something interesting that would be your next line of investigation—hint at what that might be. You can also suggest some loftier goals or ask some more questions that arise from your work.

And yes, of course, you can interpret the things that didn't go so well. Dropped a sample, added salt too quickly, lost the lottery for the best [(NH4)2SO4], you know, the average everyday annoyances.

If you are reading elsewhere, please cite that work in the of the journal Biochemistry for your references.

Next week we'll talk through a protein purification paper. If you're not entirely comfortable with the work we've done, you'll see it again. If you are fully comfortable, that'll be an easy paper to read. A win either way.

Feel free to ask questions at any point by any means.

Do remember that you only have a small picture of what it would take to fully purify peroxidase; don't be concerned that you can't give a full answer.

Since we don't have a scheduled final exam time, you'll have until the end of final exams to finish the paper. Do plan accordingly as since we're going to the end of the semester, we can't, practically, make any extensions 😉