r/Biochemistry • u/Venciczech • 2d ago

How to dissolve pepsin

Hi, I need to dissolve pepsin, either in di water or in 0,1 M HCl. But I always get a cloudy solution, with sediment after some time of sitting on the shelf. Is it normal? Articles don't mention anything specific about the process, they don't even mention activity of pepsin.

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u/BurgundyVeggies 2d ago edited 2d ago

Sigma-Aldrich's guide recommends a stock solution using 0.01 mM HCl and a concentration of 5-10 mg/mL pepsin. See here for recommendations of stock solutions and infos on activity or here for an example enzymatic assay using pepsin. Maybe it's as simple as your HCl concentration being off by a factor of 10.

EDIT: Removed an extra milli introduced by force of habit I assume.

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u/Venciczech 1d ago

Well, that's why I ask, bcs when I try to replicate certain article, they often operate with concentrations from 20 mg/ml to 40 mg/ml, and sometimes, they don't mention details, like activity, opacity of resulting stock solutions etc..

10.1016/j.jphotochem.2024.116234

10.1002/adfm.201100886

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u/BurgundyVeggies 1d ago

The main problem I'm seeing is that dry protein powder is quite an exotic state. So it's quite possible one batch will dissolve without any opaqueness and the next will put up considerable resistance to dissolve at all.
Unfortunately, I can't access the papers you mentioned but by the journals I would fear they used pepsin powder like any other chemical (so weight in is equivalent to active protein). Did you try measuring the pepsin concentration of the clear fraction (that is after centrifugation) of your previous attempts? You could also always try reaching out to the authors of the papers you mentioned asking about any solubility issues and how they solved them.

Good luck and stick to it.

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u/Venciczech 1d ago

I could try to measure the concentration of clear fraction. Also I could try to contact the authors. We will see.

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u/QSCpeptidology 13h ago

honestly some cloudiness is pretty normal with commercial pepsin powder. a lot of it isn't pure enzyme, there's insoluble junk and partly denatured protein in there. I'd just spin it down or push it through a 0.45 um filter and use the clear supernatant.

also make it fresh if you can. in 0.1 M HCl pepsin slowly digests itself, so sediment showing up after it sits on the shelf is kind of expected. keep it cold and don't vortex it hard.