The assay of chlorophylls a and b converted to their respective magnesium-rhodochlorin derivatives by extraction from recalcitrant algal cells with aqueous alkaline methanol: Prevention of allomerization with reductants

Abstract

A new method is described for assaying the difficult-to-extract (refractory) chlorophylls from so-called recalcitrant algal cells by extraction at 60°C with 2% KOH in aqueous methanol containing 1.5 mM sodium dithionite and 15% ( v v ) water. Chlorophylls a and b were converted during this extraction, by hydrolytic cleavage of both the phytyl ester bond and of the isocyclic ring, to magnesium-3 1,3 2-didehydrorhodochlorin-15-acetic acid 13,15,17-trimethyl ester and its 7 1-oxo derivative, referred to as magnesium-rhodochlorins a and b: the chlorophylls were assayed as these derivatives. The alkaline conditions may also hydrolyze the cell walls or chloroplast membranes to make these structures permeable to the extractant. Competing allomerization reactions (i.e., the oxygen-mediated cleavage of the isocyclic ring), which so readily occurs when algal or plant material is treated at room temperature with alkaline methanol, were avoided by the addition of reductants such as dithionite, ascorbate, dithiothreitol or mercaptoethanol; however, dithionite was preferred. When determining the extinction coefficients of magnesium-rhodochlorins a and b, it was found that no allomerization occurred when purified chlorophylls a and b were converted aerobically in alkaline methanol at room temperature, even in the absence of reductants. This suggests that plant and algal extracts contain allomerization-enhancing compounds. The complete extraction of refractory chlorophylls from the recalcitrant green alga, Nanochloris atomus (strain CS 183), with 2% KOH in 85% aqueous methanol containing 1.5 mM dithionite at 60°C is reported here: only 74 and 7% of the chlorophylls were extracted with methanol and buffered 80% aqueous acetone, respectively. The presence of 15% water in the alkaline methanol was also essential for total chlorophyll extraction from this green alga, but the reductant was not which indicates that heating to 60°C helps to diminish allomerization by removing dissolved oxygen from the solvents; nonetheless, reductant was still added to ensure that no allomerization occurs during manipulations prior to heating. This new procedure is recommended to replace the older alkaline pyridine extraction method for refractory chlorophylls (see Porra, R.J. and Grimme, L.H. (1974) Anal. Biochem. 57, 255–267) because the cyclic hydroxylactone derivatives of chlorophylls a and b, formed in alkaline pyridine, are less stable than magnesium-rhodochlorins a and b. Further, alkaline methanol has the advantage that it is less pungent than alkaline pyridine.