Protocol for Polyamine Analysis in Bacterial Cells:
1.Extraction & Dansylation (Derivatization):
· Take approx. 40 mg of lyophilized cells in a tube.
· Extract (Hydrolysed) with 1 ml of 0.2 M Perchloric Acid (HClO4).
· Add internal standard 1, 8-diaminooctane (25 µmol/40 mg of cells) (Required Usually for HPLC, for TLC =????)
· Incubate at 1000C for 30 min. with occasional shaking after 15 min. interval.
· After extraction the samples were centrifuged, at 8000 rpm for 10 min.
· Transfer 0.2 ml of supernatant to a tube containing 0.3 ml of Na2CO3solution (100 mg/ml) and 0.8 ml of dansylchloride solution (7.5mg/ml in acetone).
· The tube was closed tightly & dansylation was performed by incubating the tube for 20 min at 600C (usually in dark condition).
· Add 0.1 ml of proline solution (50 mg/ml) to bind excessive dansyl chloride. Incubate for 10 min. at 600C.
(Usually the volume can be adjusted as;
Supernatant (Samples) : Na2CO3 : Dansylchloride : Proline = 0.4 ml : 1.2 ml : 3.2 ml : 0.4 ml)
· Cool in refrigerator (at 4-50C)
· Add 200-500 µl of toluene and shake or vortex for 30 sec.
· Centrifuge if required to separate toluene phase from aqueous phase.
Ø Toluene Extract can be analyzed either by HPLC or TLC.
2.For TLC:
Ø The solvent cyclohexane/ethylacetate (2:3) (usually 100 ml) must be prepared some hours before chromatography & placed in tank for saturation.
(Chloform/triethylamine , 4:1 v/v may also be use as solvet)
Ø Take TLC Plate (silica gel 60,20 X 20 cm; Merck Cat. No. 105553).
Ø Samples & Standards are loaded in the application Zone by using micropipette. Generally 10-40 µl of each toluene extract are applied. Large volume must be loaded by repeated applications of small aliquots with solvent evaporation in between increment.
Ø After loading the sample, the plates are dried & then placed vertically into the chamber dipping the immersion zone into the developing solvent.
Ø The chamber is then sealed tightly.
Ø Plates are developed for 40 min. when used cyclohexane/Ethylacetate solvent & for 1 hr. 15 min. is used chloroform/triethylamine solvents.
Ø Once developed, the plate is removed from the chamber & dried quickly for 10 min. at 600C .
Ø Spots corresponding to the different amines are visualized under UV light, and identified by comparison to standards.
Ø When developed in 2:3 (V/V) cyclohexane/ethylacetate, the migration order of the different polyamines is spermine (firstly separated) followed by spermidine, and finally, putrescine and cadaverine (if present) poorly separated.
Ø When developed in 4:1 (V/V) chloroform/triethylamine, the migration order of the different polyamines is diaminopropane (firstly separated) followed by putrescine, cadaverine, and finally, spermidine and spermine.
Ø Once visualized, the spot bounders are marked with pencil.
3.For HPLC analysis:
Ø After the extraction of the dansylated polyamines, 400 ul of the organic phase (the toluene extract) is removed, dried (e.g., under a stream of ) and re-dissolved in 800ul of acetonitrile (which is compatible with the HPLC column). Finally, the samples are passes through a 0.45 pore size syringe filter before injection in HPLC.
4.Preparation of standard solutions:
Ø Stock solutions of commercial polyamine standards: diaminopropane, putrescine, cadaverine, spermidine and spermine (in the form of hydrochlorides) are prepared at the concentration of 1 mM in water (or in 0.01 N HCl).
Ø For the HPLC procedure, a stock solution 1 mM of 1,7- diaminoheptane is also prepared. The unnatural amine 1,7 diaminoheptane is used in HPLC as internal reference compound because it resolves well from derivatives of endogenous amines, elutes near amines of interest, and it is stable under storage conditions (Smith and Davies, 1987).
Ø Working standard solutions for HPLC (0.05 mM) are prepared by 1:20 dilution of the stock [i.e., 50 1 mM stock + 950 (or 0.01 N HCl)].
Ø Working standard solutions for TLC will be the 1 mM stock solutions, undiluted.
Ø Standards are stable for at least 2 months if stored at –20 °C in plastic tubes. Plastic containers (e.g., Eppendorf tubes) must be used for storage because polyamines adsorb to the surface of glass (Smith and Davies, 1987).
Dansylation of standards is carried out in the same way described for samples, but consider:
Ø Polyamine standards for HPLC are prepared by mixing 40 of each 0.05 mM working solutions (diaminopropane, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine), having a final volume of 240 Moreover, 40 µL 1,7-diaminoheptane 0.05 mM are added to each sample before dansylation, as internal reference, thus having equal experimental volumes for samples and
standards.
Ø Polyamine standards for TLC are prepared by mixing 20 of each 1 mM stock solutions (diaminopropane, putrescine, cadaverine, spermidine and spermine). Final volume of 200 (equal to sample aliquots) is achieved by adding 100 (or HCl 0.1 N). Alternatively, different concentrations of standards can be prepared (e.g., 25, 20, 15, 10 of each 1mM stock + 75, 100, 125, 150 µL water in order to obtain a standard curve for each amine (i.e.,
concentration vs. fluorescence).
Ø Dansylation must be performed in the dark since the derivatives are
light sensitive, but dim light in the laboratory is tolerable. Solution of
dansyl chloride in acetone can be stored for 24 h at 4 °C in the dark. After
the dansylation reaction, samples must be kept in the dark or in dark vials.
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| Fig:HPLC analysis of Putrescine, Spermidine & Spermine |
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Fig: Polyamine Bands in TLC |
