Comparison of Tissue Preparation Methods for Assay of Nicotinamide Coenzymes

Abstract

To prepare tissues for analysis of NAD', NADH, NADPI, and NADPH, common practice is to freeze samples in liquid nitrogen, often followed by freeze-drying, before extraction in HCI or NaOH. With cucumber (Cucumis sativus L.) cotyledons, prefreezing in liquid nitrogen or slower freezing to -20°C yielded substantially lower values for NADH and NADPH than obtained from samples homogenized immediately in acid or base. Freeze-drying after freezing in liquid nitrogen generally caused even lower values of those coenzymes. We suggest that direct extraction is more likely to yield accurate results with cotyledons and other plant parts. Nicotinamide adenine dinucleotide coenzymes are involved in functions of about 200 dehydrogenases (9). To extract these nucleotides for quantitative analysis, liquid N2 is usually used to arrest enzyme activity (1, 3, 6-9), sometimes followed by freeze-drying (6-8). While preparing cucumber cotyledons for analysis of nicotinamide coenzyme levels, we observed that immediate extraction of fresh tissues gave higher yields ofNADH, NADPH, and total nucleotides than cotyledons first frozen in liquid N2. This paper reports results of more detailed studies comparing various sample preparation methods with a direct homogeniza-tion procedure.