Measurement of Calcium
in DNA and DNP 1
The calcium content of aqueous
solutions of deoxyribonucleic acid (DNA) and deoxyribonucleoprotein (DNP) can not assayed directly because of interference effects.
An acid extraction procedure will enable direct measurements of calcium in DNA and DNP, or a standard addition technique is used at higher concentrations.
Deionised distilled water is used throughout. Glassware is acid cleaned and rinsed thoroughly in deionised water prior to use to avoid contamination.
Acid extracts are prepared by adding 1 volume of 1 M HCl to 19 volumes of sample solution containing DNA or DNP in water, and allowed to stand at 700°C overnight. Where a suspension is present, the sample is centrifuged at 2000G for 10 minutes to obtain the soluble fraction. About 99% of the DNA and about 80% of the protein becomes soluble in HCl after standing overnight in HCl. The DNA concentration of DNP is approximately 40% w/w.
The sample solution should have a concentration of DNA between 50ppm and 100ppm DNA. Therefor it should be noted that readings may cause a lack of stability if ion concentrations are present at over 100ppm. Whilst the flame photometer is able to detect ion readings over 100ppm it is not reccomended. This is due to matter which can be deposited in the nebuliser and effect further measurements by the droplets departing during other measurements. This means that the frequency that the nebuliser requires cleaning is increase and further dilution of the sample is recommended.
Standard Addition Technique
The standard addition technique is a means of estimating the concentration in a solution where interfering metals are also known to be present.
The technique measures the recovery of known quantities added to the unknown solution concentration. Sample solutions are prepared by adding 1 or 2 ml aliquots of sample solution to a calcium standard solution.
The calcium concentration of the original sample solution is calculated by plotting the concentration of standard solution, and the standard solution plus sample; the difference minus the HCl blank for each plotted curve will be the concentration of the unknown solution.
Preparation of Standard Graph
Set the flame photometer in accordance to MultiPoint/Single Ion Calibration found on page 24 of the BWB Technologies Installation and Operation Manual, to measure potassium emission. Nebulise the lithium working standard solutions and adjust the controls until steady zero and maximum readings are obtained. Nebulise the intermediate working standard solutions and construct a graph relating raw emission data (known as RAW in BWB the flame photometer) to concentration of all the standard solutions.
1 Itzhaki, R., ‘Estimation of Calcium in Deoxyribonucleoprotein and Deoxyribonucleic Acid by Flame Photometry’, Anal. Biochem. 33, (1970), p.76-86