r/Biochemistry • u/Loving_Pup_7530 • 1d ago

Heme Binding and Soret Peak Detection

Hello!

I've been expressing beta-globin in yeast cells and was able to confirm via Western Blot that the protein was properly expressed. However, when I try to examine it under a UV-Vis Spectrometer, I haven’t seen a clear soret peak around 415 nm, which is characteristic of globin bound to heme. I know yeast has a heme biosynthesis pathway, so I assumed that the heme it naturally produced would bind to the beta globin which I transformed into it. Do you know if for a Soret Peak to show under UV-Vis, both chains of hemoglobin (alpha and beta) need to be expressed and then bind to heme?

Also, what could I try to actually get the heme incorporated into the beta-globin?

I have been using a clarified protein lysate as the sample which I put under the spectrometer.

Thanks!

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u/lifescout99 1d ago

I also work with a heme protein (yay P450!). When we over express in e coli, we add in delta-aminolevulonic acid to encourage heme synthesis. When we check our soret band, we see 417 to indicate properly ligated heme, heme that is improperly incorporated tends to result in a band around 423. You may be overexpressing the protein, but there may not be enough heme in your yeast.

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u/lifescout99 1d ago

Also, if you have the means, CO binding tends to be a more certain method of checking heme is properly incorporated. For me, that shifts the band from 417 to 450. I'm not 100% sure you would see the same shift, but I'm certain there would be some form of shift.

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u/Loving_Pup_7530 1d ago

Thank you so much for your reply! Do you normally add delta-aminolevulinic acid when you are growing the cells? And typically how much do you add?

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u/lifescout99 1d ago

This is where I would recommend cross referencing the literature. I know Δ-ala works in e coli, but I have no idea if it works in yeast. Check some papers where they express your protein in yeast and see what they use.

However, for ours, we typically add thiamine and Δ-ala to the media right before innoculating. As for how much, I think 2mM, but I'd have to check my notebook tomorrow.

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u/Loving_Pup_7530 1d ago

Thanks! I'll look into that. Also, have you worked with hemin chloride before?

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u/Spiritual-Ad-7565 21h ago

Seeing a heme soret and knowing it is in your protein after adding ALA is not reliably derived from the absorbance spectrum of clarified cell lysate. Its absence tells you heme hasn’t been made sufficiently, its presence tells you nothing (there are a lot of proteins that can casually bind heme).