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HPLC

HPLC methods for recycling siderophores and separating products

Recycling HSC, dfoG and recovering dfoE (from Kadi, et al.)

Running buffer: 60% water/20% methanol/20% 0.5M acetic acid, 1 ml/min isocratic

Sample preparation: ensure protein has been precipitated and spun down/filtered out.  inject 100 uL and collect fractions or manually redirect the waste into tubes to recover.

Column: reverse phase, monitor 230 for both desferrioxamines (and everything else).  

Retention times from controls:

siderophore tR (min) detection range
HSC    
dfoG    
dfoE    
dfoB    

Separating kinetics products (adapted from Riessbrodt et al, 1990)

Running buffer: 8% acetonitrile in 0.5% acetic acid.1 ml/min isocratic note: Riessbrodt is unclear about the amount of acetic acid, but if they are using 10 mM, we would need to run 12.5% fraction as 0.5% acetic acid and the rest water and acetonitrile.

Sample preparation: dilute a 2 mM stock 1:1 with 20 mM iron (III) chloride (final concentrations 1 mM and 10 mM, respectively) and filter through a 0.22 PES filter. Inject 10 uL/run.  Ensure protein is precipitated and filtered out post-kinetics run and pre-HPLC run.

Column: reverse phase, monitor 435 nm for iron-bound complexes (ferrioxamines) and 230 for both iron-bound and not (desferrioxamines and ferrioxamines). ATP and ITC buffers will run through in flowthru, or not interfere with the visible light spectrum.

Retention times from controls:

siderophore tR (min) detection range
HSC    
dfoG 4.5 0.1 - 1.0 mM
dfoE 7.0  
adenylate    
dfoB    

[[K.Hoffmann 4/27/22]]

UV/Vis standard curves for desferrioxamines 

 

Signal at 435 nm was within the detector range for the entirety of the curve making it the best option for monitoring when not recovering substate. The equation from LINEST analysis is abs+- 0.06819 =0.98023+-0.03361(dfoG mM).
[[N.March 10/6/2021]]