141 Topics Syllabus
On Peroxidase Purification (Week 1)
Labortory icon.Lecture icon.
"It baffles me that the utterly simple and proven enzymologic approach to solving basic problems in metabolism is so commonly ignored. The precept that discrete substances and their interactions must be understood before more complex phenomena can be explained is rooted in the history of biochemistry and should by now be utterly commensensical. Robert Koch, in identifying the causative agent of an infectious disease, taught us a century ago that we must first isolate the responsible microbe from all others. Organic chemists have known even longer that we must purify and crystallize a substance to prove its identity. More recently in history, the vitamin hunters found it futile to try to discover the metabolic and nutritional roles of vitamins without having isolated each in pure form. And so with enzymes it is only by purifying enzymes that we can clearly identify each of the molecular machines responsible for a discrete metabolic operation."
-Arthur Kornberg
The core of the biochemical experimental approach to understanding living systems is purification. From purified components are functional systems rebuild. In this way, all function can be attributed to known components.
Almost experimental heaven.
Almost.
The almost, of course, it that to study purified molecules, you must first purify the molecules. Sure you can buy some common, well–studied molecules from the catalog (substitutionary atonement?), but the interesting and esoteric ones, you'll need to purify them yourself.
And thus we set off to work out a purification step (part of a larger protocol) for an enzyme. The enzyme activity we will be using today is plant peroxidase. Peroxidase catalyzes a class of reactions which reduce hydrogen peroxide (H2O2) to water (H2O) with hydrogen atoms from a phenolic donor. Conviently, the phenolytic donor we'll use today, following hydrogen donation, reacts to form a tetramer (tetraguaiacol) which is a light orange product which we can follow with the spectrophotometer (and by visual inspection). That balanced reaction is shown below.
Balanced peroxidase reaction toward guaiacol and hydrogen peroxide
Our source of enzyme will be the garden radish (Raphanus sativus). If you'd like to do this at home, and you are fresh out of radish, turnips would be a good source as well. The natural role of the enzyme is to break down reactive oxygen species and aid the plant in growth and survival.
As to the overall flow, we will homogenize radish in buffer together, each group will set out on a unique ammonium sulfate precipitation and then account for the peroxidase activity in each of the resulting fractions. In addition to the lab work this week, we'll also introduce what is in a paper (what you'll ultimately produce as a culmination of this work) and provide some themes to begin that paper. Next week we'll account the total amount of protein in each sample and put together our results into the total picture.
On to the isolation.
Extraction of Peroxidase from garden radish Raphanus sativus

Equipment:
Reagents:
  1. Remove root end, stem end and red outer layer from a fresh garden radish.
  2. Mass ≈250 g of radish.
  3. Add radish and 500 mL phophate buffer to the blender.
  4. Blend for ≈60 seconds.
  5. Divide the homogenate into 40 mL volumes in 50 mL conical tubes.
  6. Spin the homogenate at 10,000 g for 5 minutes†. This will pellet cell walls and other large debris. Pour the supernatant away from the pellet into a clean 50 mL conical tube.
  7. Collect a 1 mL aliquot of this solution. It will be our crude homogenate. Store on ice.
† This ends the corporate portion of the isolation. See Table 1 for your assignment and spin group.
Table 1. Student groups, ammonium sulfate percent saturations and first spin groups.
Lab Group  Low [(NH4)2SO4]    High [(NH4)2SO4] Spin Group
Kirk & Poole 20 25 2
Chen & Leak 70 75 1
Baksa & Farcas 90 95 1
Cramer & Hale 80 85 1
Miller & Springer 40 45 2
Anderson & Brown 30 35 2
Bianchin & Buehler 50 55 2
Hershberger & Terhune 60 65 1
Ammonium Sulfate Precipitation
One of the classic methods for protein purification of precipitation. While there are many ways in which proteins can be precipitated, ammonium sulfate tends toward being the most gentle (although, in the grand scheme of things, still rather rough). Ammonium sulfate works by taking large amounts of water to dissolve, limiting water available to dissolve proteins.
Ammonium sulfate is usually used in purifications in what is known as a "cut." In a cut, the protein solution is brought up to a particular concentration of ammonium sulfate just below where the protein of interest will precipitate. The solution is spun to precipitate proteins. The supernatant is this raised in ammonium sulfate concentration by just a few percent, causing the protein of interest to precipitate. The solution is spun again and the pellet, containing the protein of interest, is collected and resuspended.
Sounds relatively easy, eh?
Well, there is one catch.
The catch is that there is no way to reliably predict the concentration needed to precipitate a given protein. Your only choice is to do a number of studies to determine the correct concentration.
But that's where undergraduates become useful.
Ammonium Sulfate Precipitation

Equipment:
Reagents:
  1. Using Table 2 below, determine the amount of dry (NH4)2SO4 required to reach your assigned low concentration. Do note that the number in Table 2 is per milliliter of solution; scale accordingly to your ≈40 mL of crude homogenate.
  2. Mass that amount of (NH4)2SO4.
  3. Add a small amount of dry (NH4)2SO4 to your crude homogenate and swirl gently until it is dissolved.
  4. Continue adding dry (NH4)2SO4 until all your dry salt is in solution. Work slowly through this stage so that you don't have very high local concentrations as that will precipate many proteins.
  5. Rest solution on ice for 5 minutes.
  6. Spin the solution at 10,000 g for 5 minutes.
  7. Pour the supernatant into a clean conincal tube. This supernatant will continue through the purification. Dissolve the pellet in 10 mL of phosphate buffer. Label this fraction AS-I (the first ammonium sulfate fraction) and place on ice. It will not be used until next week.
  8. Using Table 2 below, determine the amount of dry (NH4)2SO4 required to raise the [(NH4)2SO4] of the supernatant to your high value (five percent higher than current).
  9. Slowly add the dry (NH4)2SO4, with swirling and rest on ice for 5 minutes.
  10. Spin the solution at 10,000 g for 5 minutes.
  11. Decant the supernatant, label as AS-III and place on ice for next week. Resuspend the pellet in 10 mL of phoshpate buffer. This is your "cut" or fraction and should be identified by the range of [(NH4)2SO4] in percentage, eg. 35-40%.
Table 2. Milligrams of solid (NH4)2SO4 to add to 1 ml of solution to achieve desired saturation at 0 oC.
Initial
Percent
Saturation
(NH4)2SO4		 Final concentration of (NH4)2SO4 in solution
  20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
0 106 134 164 194 226 258 291 326 361 398 436 476 516 559 603 630 697
5 79 108 137 166 197 229 262 296 331 368 405 444 494 526 570 615 662
10 53 81 109 139 169 200 233 266 301 337 374 412 452 493 536 581 627
15 26 54 82 111 141 172 204 237 271 316 343 381 420 460 503 547 559
20 0 25 55 83 113 143 175 207 241 276 312 349 387 427 469 512 557
25   0 27 56 84 115 146 179 211 245 280 317 365 395 436 488 522
30     0 28 56 86 117 148 181 214 249 285 323 362 402 445 488
35       0 28 57 87 118 151 194 218 254 291 329 369 410 453
40         0 29 58 89 120 153 182 212 258 296 335 376 418
45           0 29 59 90 123 156 190 226 263 302 342 383
50             0 30 60 92 125 159 194 230 268 308 348
55               0 30 61 93 127 161 197 235 273 313
60                 0 31 62 95 129 164 201 239 279
65                   0 31 63 97 132 163 205 244
70                     0 32 65 99 134 171 209
75                       0 32 66 101 137 174
80                         0 33 67 103 139
85                           0 34 68 105
90                             0 34 70
95                               0 35
On to using these samples to catalyze a reaction.
Basic catalytic assay

Equipment:
Reagents:
The basic assay follows this pattern:
In a cuvette:
  1. Add 2860 μL of phosphate buffer
  2. Add 60 μL of 0.1 M H2O2
  3. Add 60 μL 1% guaiacol
  4. Add 20 μL radish extract fraction
As you add the radish extract fraction, mix the tube and start the clock. After sixty seconds, record the absorbance at 460 nm.
Alright, that's the basic assay, but what samples do we measure, how many times and how do we handle our data?
Perform the above assay on each of the following samples, in turn.
  1. Crude homogenate
  2. Crude homogenate
  3. Crude homogenate
  4. (NH4)2SO4 cut
  5. (NH4)2SO4 cut
  6. (NH4)2SO4 cut
Yes, do two reactions, three times each. The reason (I know you're wondering) is so that we can report a mean activity with a standard deviation. When we take samples and build reactions, there will be a bit of random variation. The mean gives us the average value, and the standard deviation gives us a feel for how tightly those values cluster around the mean.
For example, if you had taken an exam and asked me what I thought you had scored, I could say 80±5, and you'd be pretty confident that you did well. If I said 80±15, you'd be a little more nervous. You could have made an A, or you could have barely passed. A pretty wide swing.
And now on to analyzing these data.
Calculating Units
Wow, a whole section on units. That's surprising. Must be alot about units.
Actually, it's all about U, the units of activity.
Instead of reporting activity in units like μmol product per minute per milliliter of solution, biochemists will define a complex set of units, with a magnitude, and simply call it one unit (U) of activity. It makes tables smaller, equations clearer and generally makes for neater papers and accounting all around.
We will define one unit of peroxidase activity as 1 x 10-10 moles of guaiacol reacted per minute per milliliter of enzyme solution. A mouthful. Written more succintly:
$$1~U~:=~1~x~10^{-10}~{moles~guaiacol\over{minute\cdot mL}}$$
Well, that's neater than I was expecting. What's the catch?
The catch is that our measurement is absorbance, not concentration, time or volume. The pathway of conversion is long, but not overly convoluted.
The first step is to convert absorbance to concentration using the Beer–Lambert Law:
$$A=\epsilon l c$$
Where A is the absorbance, ε is the extinction coefficient (26,600 M-1cm-1 at 470 nm for tetraguaiacol), l is the pathlength of the cell (1 cm) and c is the concentration. This equation is used to convert your measured absorbance to a concentration of tetraguaiacol.
To convert from the concentration of tetraguaiacol to the moles of guaiacol which reacted, it's a bit of dimensional analysis involving the stoichiometry (1 tetraguaiacol to 4 guaiacol) and the total volume of the reaction (3 mL). That conversion will look like:
$${??~moles~tetraguaiacol \over 1~L} \times {4~moles~guaiacol \over 1~mole~tetraquaiacol} \times {1~L \over 1000~mL} \times {3~mL \over } $$
The completion of our unit calculation will involve dividing the moles of guaiacol by the time of the reaction in minutes (1 minute) and the volume of enzyme solution used in the assay (0.020 mL). That will put us in units of moles guaiacol per minute per milliliter. All that is left is to divide by 1 x 10-10 to convert that value into units of Units, U.
See, not so bad.
About that mean and standard deviation, convert all three values for the crude homogenate to U, take the average and then calculate the standard deviation according to:
$$1 \sigma = \sqrt{\sum{(x - \bar x)^2} \over (n)}$$
Where x is the value, x bar is the mean value of the variable (the average) and n is the number of measurements (three for us).
Report mean ± 1σ for the crude homogenate.
Repeat the analysis for your (NH4)2SO4 cut.
What to Save for Next Week
These samples can be labeled (markers write well directly on tubes, there is labeling tape to the right of the board), placed in a multitube rack and placed in the cooler for the week. A low shelf to the right should be open.
Lab Report
The only work you need to submit for this week is the mean and standard deviation of your crude homogenate and your (NH4)2SO4 cut. It's ungraded, but since we're working together, we'll need the these numbers for next week for a complete dataset.
But, the ultimate goal is to write a bit of a paper based on our work. There are some things you can start on, if so inclined.
Title
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).
Abstract
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.
Introduction
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 is quoted at the top, 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 even. 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.
Methods
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 and the measurement of activity. You can be brief here since all of the methods are above.
Results
This section has the data, largely in figures and tables with enough prose to connect it all together. Our major result will be a large table, but we'll need the data from next week to complete that table. More to come.
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.
Discussion
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). While you might not have a good reason as to why, the composition of ionziable groups (DEHKR, in single letter codes) may suggest something about solubility.
Last updated 03 April, 2025.
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