Economical synthesis of 14C-labeled aminolevulinic acid for specific in situ labeling of plant tetrapyrroles.

Abstract

The application of metabolic radiolabeling techniques to plant tetrapyrroles, i.e., chlorophyll and hemes, is complicated by the difficulty of obtaining sufficient quantities of radiolabeled aminolevulinic acid (ALA). ALA, the first committed intermediate in the tetrapyrrole biosynthetic pathway, is inconvenient to synthesize chemically and is generally not produced in significant quantities in biological systems. Radiolabeled ALA is therefore usually quite expensive and available only in limited quantities. Here, we describe bulk biosynthesis and purification of 14C-labeled ALA from 14C glycine. We first cloned ALA synthase (ALAS) from Rhodobacter sphaeroides into an expression vector for expression and purification as a fusion with maltose-binding protein. We then used the purified ALAS to synthesize ALA in vitro from 14C-labeled glycine and succinyl-coenzyme A. Finally, we used ion exchange chromatography to separate the ALA product from the crude reaction. We achieved conversion and recovery efficiencies of 80-90%, and chlorophyll radiolabeling experiments with the 14C ALA product revealed no detectable non-specific incorporation into proteins. The ability to economically produce robust quantities of 14C ALA using common methodologies provides a new tool for working with tetrapyrroles, which includes both hemes and chlorophylls and their respective binding proteins. This tool allows the specific detection and quantification of the tetrapyrrole of interest from standard acrylamide gels or hybridization transfer membranes via radiographic imaging, which enables a wide array of experiments involving spatial and temporal resolution of the movement of pigments as they are synthesized, incorporated into their target binding proteins, and eventually degraded.