The determination of heme b in marine phyto- and bacterioplankton

A method for the quantification of heme b in marine phyto- and bacterioplankton is described. Heme b was extracted from filtered cells using a solution of 2.5% octyl β-glucopyranoside in 0.02 M ammonium hydroxide. The extract was analysed by high performance liquid chromatography diode array spectrophotometry. Maximum absorbance for heme b was at 400 nm. Heme b was separated from other pigments using a polystyrene divinyl benzene stationary phase and a gradient elution programme with 0.1% (v:v) nonafluoropentanoic acid in water and 50:50 (v:v) isopropanol:acetonitrile as the mobile phases. Heme b was quantified using Fe (III) protoporphyrin IX chloride (hemin) standards. The detection limit, calculated from 3 × s.d. of the lowest standard was 0.08 pmol or 1.57 nM with a 50 μL injection volume. The first data for heme b in marine phyto- and bacterioplankton are reported. Heme b contents are reported for the eukaryotes Thalassiosira weissflogii, Thalassiosira oceanica, Dunaliella tertiolecta and Emiliania huxleyi, and the prokaryotes Synechococcus WH8102, WH7803, RCC307, Erythrobacter litoralis, Roseobacter denitrificans and Vibrio natriegens. For T. weissflogii, T. oceanica, D. tertiolecta and E. huxleyi cellular heme b concentrations varied between 12 and 60 μmol L− 1 and chlorophyll a to heme b ratios varied between 216 and 309.