Problems: RP-HPLC sheep hemoglobin

[sjl]

Hi there,

Apparently the conditions of RP-HPLC (Acetonitrile, TFA) should cause dissociation and de-heme hemoglobin subunits. However, i have optimised the gradient and carried out a long run time and can still not see individual peaks for the alpha and beta chains. I can only see one protein peak plus a heme peak.
However, if i carry out an acid acetone precipitation on hemoglobin and then inject into the column, i can get these two subunits. Is this to do with the column i am using or something, i have no idea?
Can you tell what peak is the alpha and what peak is the beta without mass spect??

Another reason i think the conditions of HPLC havent dissociated and de-hemed sheep hemoglobin:
I digested hemoglobin with pepsin over 24hrs. When each of these time point samples are injected into the column, the hemoglobin peak decreases as it is digested, but the heme peak continually increases over 24hrs. If the hemoglobin were dissociated/de-hemed by the HPLC conditions at the beginning, the heme peak should be a constant size over the 24hrs.

Im sooo puzzled..please help!!!!!!!

Oh i have tried two columns:
Phenomenex Jupiter 4u Proteo 90A, 250mm x 4.6mm C12
Phenomenex Jupiter 10u proteo 90A 250x10mm C12

[sjl]

anyone? any ideas i would be grateful for

[Andy Alpert]

It takes a finite amount of time for reversed-phase (RP) conditions to dissociate the globin chains in hemoglobin. It's possible that the kinetics of dissociation are slower for sheep hemoglobin than for other mammalian hemoglobins that have been analyzed this way. That would be consistent with your observations. Is there any literature on the relative kinetics of dissociation of hemoglobin from different animals?

Can you determine which globin chain is which without mass spectrometry? Sure! Alternatives:
1) Run an authentic standard of either the alpha- or beta- globin chain of sheep hemoglobin and compare the retention time with your sample's chromatogram. The chances of your obtaining a standard are so low that you may consider this suggestion to be facetious;
2) Edman sequencing, either of the intact globin chain or of a fragment after digestion. Mass spec is a lot more convenient if it's an option.

May I ask what the objective is here? In general, intact hemoglobins are analyzed by cation-exchange chromatography. This can also separate the dissociated globin chains from each other, although most labs use RP because it's convenient for them to do so and because it's always been done that way. Incidentally, what kind of RP column are you using? Most labs use C4 material for this separation and for large polypeptides in general.

[sjl]

Hi thanks for replying

I have read one article that says the sheep hemoglobin tetramer is quite stable in terms of disociation, and that urea denatures most hemoglobins with the exception of dog and sheep. However, none of the other articles on sheep hemoglobin mention anything like this. Infact i have seen RP-HPLC chromatograms of sheep hemoglobin separated into alpha, beta and heme (using the same conditions as me)-this confuses me as i cannot obtain them. If i carry out an acid acetone precipitation and then run RP-HPLC i seem to get the separation. In terms of the identity of each peak, can i assume they they are in the same order as mentioned in literature or is this column dependent?
Would the column have a huge influence on not being able to see the individual alpha and beta peaks?? I have seen in literature, as you said, most labs use a C4 column for this separation, however i have used C12, as thats what we have available.
i have tried two columns:
Phenomenex Jupiter 4u Proteo 90A, 250mm x 4.6mm C12
Phenomenex Jupiter 10u proteo 90A 250x10mm C12

Also, in literature, the heme peak elutes before the hemoglobin chains, whereas in my case, the heme elutes after the hemoglobin peak. Is this also due to column type?

My project is looking at antimicrobial properties of hemoglobin. I am testing the antimicrobial activity of:
1) native hemoglobin
2) its heme-free subunits i.e. the globin chains (can't seem to obtain these by HPLC alone although they do in literature)-semiprep column
3) peptides derived from digesting hemoglobin with pepsin-analytical column

THANKS!!!!!!

[Andy Alpert]

Once again, SJL:

If you want to get the results in the literature, then you must use the methods that were used in the literature. Get a C4 column! You will then get the same order of elution of heme group and globin chains that were published in the literature, permitting you to identify them without using standards or more involved instrumental analysis.

It is apparent that your failure to see the different globin chains is a function of the method used to prepare them, since you say that you do see separation with your C-12 column if you use a more involved denaturation procedure. I presume that the papers in the literature that deal with sheep globin chain analysis used more involved processes for their denaturation than you have done, although they may not have described all the details in the papers. Try something like incubating the hemoglobin with 100 mM hexafluoro-2-propanol (HFIP) for an hour prior to running it on the C-4 column. That shouldn't affect the chromatography significantly. The denaturing effects of organic solvents and of chaotropes (e.g.; HFIP; urea; NaClO4; etc.) are additive. You could even include 50 mM HFIP in your mobile phases if necessary.