Since nobody replied and I have studied this subject pretty extensively in the past year I will answer it myself so other people can benefit from this knowledge:

Heme refers to molecules in which iron is in it's reduced (Fe2+) state. It is usually reduced by a protein it is attached to. See, in the body, heme must be bound to, most commonly a protein (hemoglobin, cytochrome, transporter, etc). Once heme is not bound to a chaparone it must be oxidized immediately, or it can be toxic to the cell in it's reduced state. It is not toxic when bound to a protein since they share electrons. Once heme is by itself it is immediately oxidized to it's Fe3+ state, and in this state it is termed "hemin". 

When measuring heme levels florescently heme must be separated from it's carrier, causing ixidation and resulting hemin. The consequence of this is that unless you have a very specific assay that can detect heme when it is bound to a protein (which I am unaware of the existence of such assay), you cannot measure levels of heme - but only hemin. Nevertheless, since all heme converts to hemin anyways, and level of cellular endogenous hemin is extremely small - measuring hemin is as good as measuring total heme.

So if you wanted to.measure levels of heme that is bound to a specific protein you would need to first isolate the protein, and then measure hemin levels in that protein. Same for specific  tissue or cellular compartment.

Hope this helps.if not, I am always available for follow up questions

Since nobody replied and I have studied this subject pretty extensively in the past year I will answer it myself so other people can benefit from this knowledge:

Heme refers to molecules in which iron is in it's reduced (Fe2+) state. It is usually reduced by a protein it is attached to. See, in the body, heme must be bound to, most commonly a protein (hemoglobin, cytochrome, transporter, etc). Once heme is not bound to a chaparone it must be oxidized immediately, or it can be toxic to the cell in it's reduced state. It is not toxic when bound to a protein since they share electrons. Once heme is by itself it is immediately oxidized to it's Fe3+ state, and in this state it is termed "hemin". 

When measuring heme levels florescently heme must be separated from it's carrier, causing ixidation and resulting hemin. The consequence of this is that unless you have a very specific assay that can detect heme when it is bound to a protein (which I am unaware of the existence of such assay), you cannot measure levels of heme - but only hemin. Nevertheless, since all heme converts to hemin anyways, and level of cellular endogenous hemin is extremely small - measuring hemin is as good as measuring total heme.

So if you wanted to.measure levels of heme that is bound to a specific protein you would need to first isolate the protein, and then measure hemin levels in that protein. Same for specific  tissue or cellular compartment.

Hope this helps.if not, I am always available for follow up questions

Dear Lee,

thank you for posting about this. which kit are you using to measure Hemin?

Hi Mohammad, I do not use a kit. Our lab is all about doing stuff ourselves and not using kits. For good and for bad. I use either of these two methods:

1. http://smolkelab.weebly.com/heme-measurements.html

2. https://www.ncbi.nlm.nih.gov/pubmed/20954156

I will soon publish my own data and the exact method I have used will be described there. In the meantime if you need any specific help you can contact me directly.

Good luck.

Shalom lee,

grat post! you helped a lot!

I am currently struggling with hemin toxicity in cell based experiments. Indeed as you mentioned above, hemin is quite toxic! so i tried mixing it with BSA before adding it to the cells but it did not have the seme effect as expected (or any effect at all).

do you have any experience in adding hemin to cells?

Hi Ala, 

Hemin is only toxic in specific concentrations. What did you base your quantities on? There are some articles about it I can search it for you when I have a moment.  

Also, when trying to add hemin without BSA did the cell die? Or you didn't try adding it by itself? Let's continue the discussion via email, it will be easier, contact me at [email protected]

Thanks lee 

i will contact you soon!

what is the % of binding hemin to albumin