Separation of double-stranded DNA in conventional and isoelectric buffers: studies on stability and separation performance.
In the capillary electrophoresis of double-stranded DNA in isoelectric buffers, worsening of resolution was observed in electropherograms as a function of time passed from the preparation of the separation solution, which consisted of 0.7% hydroxypropylcellulose, HPC, Mr 10(6), diluted in 150 mM histidine buffer. The DNA standards used were: kilobase pair-ladder, Marker V and Marker VI. In order to understand what happens in the histidine-HPC solution with ageing, the absorbance spectrum (200-500 nm), the conductivity and the pH of the solutions as a function of time were monitored. Fresh His gave a distinct peak at 206 nm. For all the solutions a significant diminution in the maximum absorbance value at 206 nm was observed as a function of ageing, with the concomitant appearance of a peak at 278 nm as the solutions became older. Also the conductivity increases dramatically with the ageing of the solutions and seemed to reach a plateau after ca. 40 days. In concomitance with the conductivity increments with time, the pH of the His solution (isoelectric point, pI=7.6) grew slowly up to pH 7.9; these combined data indicated that a new species contributing to the conductivity and altering the pH was formed from the His molecule, suggesting that His degraded in time. When the dipeptide His-Gly was used instead, a similar ageing phenomenon was observed, but with much reduced kinetics. Mass spectrometry, coupled to RP-HPLC, detected, in aged His solutions, in addition to intact His, two main degradation products: a 110.1 u species and a 93.2 u compound. The mass of the former coincides with the protonated species derived from the formation of a Schiff base on the alpha-amino group of His and subsequent decarboxylation without transformation of the final Schiff base into a chetonic group (a histamine-like molecule terminating with an imino, rather than with an amino group).
Magnusdottir
S
S
University of Verona, Department of Agricultural and Industrial Biotechnologies, Italy.
Gelfi
C
C
Hamdan
M
M
Righetti
P G
PG
D002021:Buffers; D002853:Chromatography, Liquid; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry
D002021:Buffers; D004247:DNA