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I am trying to buy 1g of horseradish peroxidase. On sigma-aldrich, it is sold as units/mg. I am confused as to what this unit is. It comes in 50-150, 150-250, and 250-330 u/mg. The way I currently understand it is if I buy 25 KU (kilo-units?) of 50-150 u/mg, than I should have at minimum 167 mg of the peroxidase. Is this a correct understanding? The price of 25 KU of horseradish peroxidase is $95, so it seems ridiculous to pay that for only 1 2/3g.

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Sigma Aldrich defines their units as:

One pyrogallol unit will form 1.0 mg purpurogallin from pyrogallol in 20 sec at pH 6.0 at 20 °C.

Assuming you're doing a biological imaging experiment, these activity values tell you much more about what you're getting than mass.

From a casual internet search it appears that HRP is pretty expensive. I would consider whether you actually need so much of it for your research.

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  • $\begingroup$ Indeed. 1 g is a lot of HRP for most things. 10 mg of ~150 U/mg HRP is enough to do several well plates' worth of peroxide assays for me. I usually aliquot out a stock solution and freeze the extras. $\endgroup$ – Michael DM Dryden Feb 15 '15 at 1:46
  • $\begingroup$ I am using this for synthesis of polypyrrole. To do so, I need to be able to measure out masses of HRP. $\endgroup$ – dfghj Feb 15 '15 at 2:45
  • $\begingroup$ @jarred: no, you probably don't. What you need to know is the activity of a certain amount of enzyme, which is what U/mg gives you. $\endgroup$ – Gerhard Feb 15 '15 at 12:23
  • $\begingroup$ And I hope you are aware that you will have to work in aqueous buffer solution when using enzymes, which might be a bit limiting when working with pyrroles. $\endgroup$ – Gerhard Feb 15 '15 at 12:30
  • $\begingroup$ If you're talking about this paper, they use 1 mg of ~170 U/mg HRP in 10 mL of solution (probably because they didn't want to do serial dilutions and 1 mg is about the lowest that's practical to weigh out), so you just need to make up a solution that's around 17 U/mL. How much HRP you need depends on the activity level of the stuff you buy. $\endgroup$ – Michael DM Dryden Feb 15 '15 at 18:44
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The enzyme is sold by activity, not by weight. The different descriptions from Sigma refer to the amount of active enzyme (in units according to their definition) in 1 mg of the powder or lyophilized residue they sell you. You will not be buying pure HRP. "Pure" HRP is a bit of a misnomer anyway, since it comes it at least seven isoforms, differing slightly in MW and glycosylation patterns, etc. So if the U/mg number is higher, the amount of active enzyme per mg of residue is higher.

Most HRPs seem to have a molecular weight of around 40,000 g/mol. If you knew the rate constant $k_{cat}$ of HRP for your substrate in your assay conditions, you could use the rate constant and the MW to calculate a "theoretical" activity in terms of U/mg. My guess would be that this theoretical activity would be higher even than 250 - 330 U/mg.

The ratio of the U/mg that you buy to this "theoretical" activity is the weight fraction of what you buy that is active enzyme. Probably for the cheapest preparation, more than 90% of the weight is not active enzyme. There are probably salts, water, and inactive/misfolded HRP molecules, and other protein contaminants in there to varying degrees.

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