The Iron and Heme Core’s primary mission is to facilitate research into the role of heme, heme precursors and transition metals in both normal and disease states. The iron and heme core lab has extensive experience with the separation and identification of tetrapyrroles and with running and developing heme biosynthesis pathway enzyme assays. We are offering the following services:
UPLC Analysis of Total Heme and PPIX
A Waters Acquity ultra performance liquid chromatography (UPLC) system equipped with a reverse phase C18 column, a photodiode array detector and a fluorescence detector is used to determine total heme and protoporphyrin IX (PPIX). Samples are subjected to solvent extraction and all noncovalently associated heme and PPIX are transferred into the organic phase and quantified. Hemin chloride (the Fe+3 equivalent of heme formed during sample processing) and PPIX are used as standards for quantitation.
While PPIX may be at extremely low to undetectable levels in normal unperturbed biological samples (due to conversion to heme), PPIX and ZnPPIX are detectable (in our standard analysis) in perturbed systems such as in MEL cells induced to differentiate, and in liver samples of mice with ALA in the drinking water. Ferrochelatase is known to catalyze the insertion of other divalent ions, mainly zinc, into protoporphyrin IX, hence the observation of elevated ZnPPIX accompanying PPIX concentration that is raised under certain experimental conditions.
Spectral Analysis of Heme
An Agilent 8453 diode array spectrophotometer can also be used for the spectrophotometric analysis of hemes. Typically, samples are solubilized in 200 mM NaOH in the presence of pyridine to obtain the pyridine-ferrohemochrome derivative spectra. Simultaneous determination of hemes a, b, and o, is possible from the pyridine hemochrome spectra. Each heme species is quantified using the well-established alpha band extinction coefficients at characteristic absorbance maxima obtained from the reduced-minus-oxidized spectra.
reproduced with permission of themedicalbiochemistrypage, LLC
Porphyrin Analysis (UPLC)
Porphyrins (excluding protporphyrinogen IX (PPIX) which is assayed together with heme) from urine, feces, cell lysates, serum, blood, media concentrates from mammalian cells and yeast and other biologic samples are analyzed using a Waters UPLC system. Porphyrins are separated on a reverse-phase C18 column, 2.1x 100 mm BEH, and visualized with the tandem high sensitivity photodiode array and fluorescence detectors of the UPLC.
Molecules analyzed include: the biosynthetic intermediate porphyrins: uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin. (Standards are available from Frontier Scientific, Logan, UT.)
Porphyrin Isomer Analysis (UPLC)
Uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin of both I and III isomers will be assayed with this selection
We may also assay for d-aminolevulinic acid (ALA), bilirubin, biliverdin and nonstandard porphyrins where a standard is available
Assays of Heme Biosynthetic Enzymes
Activity may be measured for the following enzymes:
ALAS 5-aminolevulinic acid synthase
ALAD/PBGS aminolevulinate dehydratase/porphobilinogen synthase
PBGD porphobilinogen deaminase
U3S uroporphyrinogen III synthase
UROD uroporphyrinogen decarboxylase
COPOX coproporphyrinogen III oxidase
PPOX protoporphyrinogen IX oxidase
FECH ferrochelatase
Protocols for these enzyme assays are available for download through the Shared Resources – Protocols tab in this website [link]
reproduced with permission of themedicalbiochemistrypage, LLC
Analytical Service – Metals analysis ICP-MS
An Agilent 7900-ICP mass spectrometer is available for the determination of metals, such as Fe, in biological samples.
For this service, we would like 30-100 mg of sample, sent frozen, and will prepare the sample for metal analysis here. For cells, please wash the pellet, and remove liquid prior to freezing. The media or wash buffer can be a possible source of metal contamination. Please also send us a sample of your wash buffer to test. If you are able to use metal-free or trace metal materials/reagents that is preferable. If you choose to send us homogenates, please prepare the homogenate in pure, metal-free (trace) water, and send a matching control (containing homogenization water).
Please let us know your preferred method of sample normalization. We typically normalize samples to protein concentration after running a BCA protein assay on the homogenate. For tissue, we can also weigh the sample. If you wish the sample to be normalized to cell number, please count the cells prior to sending and let us know how many cells in the pellet.
If you wish to test a metal other than Fe, Mn, Zn, & Cu, please contact us first to discuss.
ICP -MS Useful Information:
Instrument: Agilent 7900 ICP mass spectrometer
Sample digestion: Our core digests biological samples using Nitric Acid, Hydrogen Peroxide and heat (hot block method). We digest small samples in polypropylene tubes, heat them until dry and resuspend in 2% Nitric Acid for ICP-MS analysis. We utilize Agilent certified multi-element calibration standards to quantify your element of interest.
The metals that we most commonly measure are: Fe, Cu, Zn, Mn
Our users also request: Ca, Mg, Ni, K, Rb, P, Co, Cd
We have the following multi-element ICP-MS calibration standard solutions in stock:
Agilent Standard2A: Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Tl, U, V, Zn
Agilent Environmental Standard: Ca, Fe, K, Mg, Na, Ag, Al, As, Ba, Be, Se, Th, Tl, U, V, Zn
Agilent Multi-Element Calibration Standard-4: B, Ge, Mo, Nb, P, Re, S, Si, Ta, Ti, W, Zr
We request a minimum of 20 mg of tissue (we prefer at least 30 mg), 5 million washed mammalian cells, or 100 uL of washed, packed pellet for yeast or bacteria. Amount of material required will differ between cell types, please contact us for more information. Please remove all buffer prior to sending us cell pellets. Too much PBS or water can dilute digestion. Please keep packed cell volume to less than 100 uL if you wish to digest directly and normalize to cell number. Please send us a sample of your wash buffer to allow us to subtract background.
For any type of experiment please send us multiple control samples (buffer) so that we can subtract background. These are free.
We can measure several elements at once (no increase in price if the elements are in the same calibration standard). We do need for you to let us know each time (by Email), at the time that you submit your samples, which elements that you are interested in. The elements of interest must be entered into the software prior to the ICP-MS run for a quantitative measurement.
The instrument also does a qualitative element scan (all elements) in each run. Feel free to ask us for this qualitative data if you have an interest.
We are able to homogenize tissue here using Ceramic Bead Tubes.
We recommend biological replicates for each sample type. Please let us know each time you submit your samples if you want us to quantify protein for you, for normalization purposes. We use a Pierce BCA Protein Assay for protein quantification. We typically lyse the sample for the protein assay as follows (bead tubes and water for tissue, add water and sonicate for cells, add water and freeze-thaw for mitochondria). You can also do this in your own lab (including protein quantification) and bring us the remaining lysed sample. Please only use ultra-pure water or PBS and bring us > 1 mL of the water/buffer that you used in your sample preparation. Iron can be found in just about everything so washes, and choice of buffer can impact signal to noise. Since each experiment is different, we are happy to speak with you to help you to prepare your samples.
If you are shipping the sample, please ship the sample frozen. If you deliver the sample and want protein to be quantified here for you, please bring the sample to us cold or preferably frozen.
Please see pricing tab for information on charge breakdown. We typically use the Optima Nitric Acid in our digests and runs.
Additional Information:
Please allow at least two weeks for service completion.
Heme and porphyrin analysis will yield data in picomoles per mg sample protein.
Heme biosynthesis assays will yield data in nanomoles per mg sample protein per hour.
