The peptide I am measuring is 30 residues long, and analogues will be lipidated at several different locations. We are currently worried that lipidation (with palmitate) will affect aqueous solubility of the purified peptide. Obviously there are crude methods at testing solubility e.g. visually/ by measuring acidic/basic residues/ UV spectrophotometer/ nephelometry.
I feel there must be a better way to accurately quantify the limit of solubility for these peptides but I don't exactly know what or how. I've considered using BCA/ Bradford assay to quantify peptides dissolved, but these are not ideal as well (due to the size of the peptides, standard used etc). Any suggestions are welcomed.
P.S. Purified peptides will then be tested in vitro in a cell-based assay followed by in vivo in mice. It is easy to inspect the solution when doing the in vitro experiments, but it's crucial for me to determine solubility to make sure peptides are still in solution once administered to the mice.
The solubility of your modified peptide will depend on lipid content as well as the nature of peptide - pH an temperature may also effect the solubility.
With multiple palmitate groups, chances are slim that you will have high solubility in aqueous solutions.
Solubility of your modified peptides in aqueous medium and its bioavailability to the target cells in vivo are not always directly related. As you probably already know, there are many plasma proteins that interact and bind with lipophilic compounds, and deliver them preferentially to certain cells (for example, liver and kidney).
Depending on how poorly soluble in water based solutions/buffers is your modified peptide, you should consider alternative methods to stabilize the suspension or use solvents such as DMSO. if you can dissolve it at high concentration and your working concentration is 3 orders or magnitude lower, you may be able to use such methods, assuming that once diluted, even if not completely solute, it will complex with plasma proteins and will remain in circulation.
Thanks Mr Alam for your insight. I will consider DMSO as a solvent (or at least have a small percentage of DMSO in the solvent). pH and temperature will be various factors I will also have to consider to ensure solubility across the dual experimental formats. And once we have determined positive activity in a pilot study in vivo, we will proceed with a much larger scale experiment to get the pharmacokinetic profile.
There will only be a single palmitate group per peptide analogue, not multiple lipidation per peptide.
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