Quantitative separation of nucleotides on mercurated dextran

Area of Science:

• Biochemistry
• Chromatography
• Analytical Chemistry

Background:

• Dextran gels like Sephadex are widely used in chromatography.
• Chemical modification of stationary phases can enhance separation capabilities.
• Nucleotide separation is crucial for molecular biology and diagnostics.

Purpose of the Study:

• To develop a novel method for separating nucleotide mixtures.
• To investigate the efficacy of mercurated Sephadex G-25 in nucleotide fractionation.
• To understand the underlying principles of separation based on organomercurial interactions.

Main Methods:

• Chemical modification of Sephadex G-25 with monofunctional mercury.
• Fractionation of mono- and dinucleotide mixtures using mercurated Sephadex G-25.
• Elution with Tris-bicarbonate/carbonate buffer systems at varying pH or with competing ligands like chloride ions.
• Analysis of separation behavior through retention volume calculations and comparison with known interaction parameters.

Main Results:

• Mercurated Sephadex G-25 successfully separated mixtures of four mono- and four dinucleotides into eight distinct fractions.
• Complete separation was achieved for mixtures of adenosine phosphates (mono-, di-, tri-), demonstrating broad applicability.
• Fractionation was effective using both gradient pH elution and constant pH elution with competing ligands.
• Experimental results aligned with theoretical predictions based on mercury-ligand complexation.

Conclusions:

• Mercurated Sephadex G-25 is a highly effective stationary phase for nucleotide separation.
• The separation mechanism is governed by the affinity of nucleotide nitrogenous bases for organomercurial compounds.
• This method offers a robust approach for analyzing complex nucleotide mixtures.