Chromatography Forum

Hi,

I am new here, im a masters student in NZ. I am testing the antimicrobial activity of peptides from hemoglobin.
The hemoglobin was isolated from whole blood using ammonium chloride (lysis). I then diluted the hemoglobin solution with 0.1M sodium acetate (ph 4.5) and digested the hemoglobin with pepsin in the presence of urea (5M). At the appropriate time point i stopped the reaction by raising the ph with di-sodium tetraborate (0.32M).
I then freeze dried the sample and want to collect all the peaks using RP-HPLC (there are many peaks, approx 50).
The problem is that the two HPLC's in my lab will only allow me to load a max of 250ul. This would be ok if i could get my sample more concentrated, but if i get it concentrated the sample becomes too viscous (even though the protein concentration is still low). Currently i can't seem to get the conc much higher than 2mg/ml protein without it being too viscous. The pressure in the HPLC gets very large and my run stops (my sample has been filtered). I need the sample concentrated, because the amount of protein injected is going to be divided by a large number of peaks, each are going to be collected and tested for antimicrobial activity.
Should i remove the salt somehow or? One of the lab tech's said they have a very old semi-prep HPLC and they are currently 'trying' to set it up for me. I could then inject up to 2ml of sample. I have both analytical and semi-prep columns available. All the articles on my topic dont mention removing salts, and they use semi-prep hplc.

What do you suggest??

Thanks,

Sarah

I would be grateful for any help.

Cheers!

if the viscosity is too high due to the salt then:
one friend of mine from a biotech company had a great idea for that
he took very old "dead" SEC/GPC columns and he started using them to separate the salts from the protein with 100% water.
a kind of crude and simple SPE. he collects the compounds, the first ugly peak on the column.
then he freeze-dries.
that way he can increase loading.

he even repacks back to smaller lenght columns of 150 or 100mm because then he can use the media for longer time and gets faster, so if you need to order a new column save money and get a short 50-100mm column lenght only

but could it be due to the nature of your sample?
do you have an idea of the sizes of your peptides?
what is there nature of them?
50 peaks means a lot of compounds, are you getting some of them stuck on the column maybe?
can you give more details of the separeation method used?
mobile phase, column, temp?

and still i wonder, a peptide map with 50 peaks, how much can you really get more loading before you lose separation between the different peaks?
can you post a chromatogram?

Hello.
Firstly for me as a paramedic with a quarter-century of experience and a pharmacist with 20 years experience of the country itself formulation of the problem.
Many pathogenic hemolyzing bacteria grow well on red blood agar and calmly eat hemoglobin.
Where did the idea that hemoglobin is not for bacteria breeding ground, but the poison? I'm afraid that when the original formulation of research problems was made a fatal error.
However you have the opportunity to test this.

With regard to this problem.
1) Please note that hemoglobin non stability to oxidation.
2) Please note that the hemoglobin (and in general all the components of blood) can be a breeding ground for bacteria.
3) Keep in mind that we need a thorough cleaning of all proteins - the same pepsin really capable of lysing bacteria, and in blood plasma contains antibodies , lysozyme and complement system.

How would I have done.
1) Before allocated to hemolysis of erythrocytes gematokrinnoy centrifuge, separating them from the plasma and partly from leukocytes ..
2) destroying red blood cells would not pepsin, and by osmotic shock. You are fully press the plasma in a centrifuge, dilute the red blood cells in hypertonic salt solution (3-5%), soak an hour and then rapidly pour the red blood cells in distilled sterile water, preferably hot or icy cold front (this will ensure sterility during the operation and remove oxygen).
As the water will be absorbed by osmosis into the red blood cells - they will swell, turning into balls. And eventually some of them burst. Usually when this is added EDTA, but you do not - it takes iron from heme.
You can then centrifuged destruction of erythrocytes from an aqueous solution of hemoglobin.
3) For the separation of these proteins previously used ion-pair chromatography on gel-type Dowex sorbents. You can use the free-flow glass columns of large volume, for example to make the column axial compression of a bottle of champagne (she cut off the bottom). Consult with older professionals.
4) Do you suffer from too low a concentration? - Water can boil down freeze-drying. This is standard equipment, which certainly has a microbiologists. But I'm not sure that the hemoglobin will be crystallized into a powder.