An assay for the measurement of the protein content of cells immobilized in carrageenan

Abstract

A reliable and reproducible method for the estimation of the protein content of fungal cells immobilized in a carrageenan gel is described. The procedure depends upon the acid lability of the polysaccharide gel at 90 °C and on the acetone solubility of accumulated phenolics. Freeze-dried gel beads (2–3 mm) containing entrapped cells of Penicillium urticae were ground to a fine powder and samples of powder (~20 mg) were sequentially extracted with hot 1 N HCl – 0.9% NaCl and acetone. The precipitated residue contained the cell protein, which was then solubilized with 1 N NaOH at 90 °C and quantitated by the Folin–Lowry method. Interferences from both carrageenan and phenols were thus eliminated. The presence of carrageenan (20–25 mg) did not affect the recovery of varying amounts (0–2500 μg) of bovine serum albumin. The recovery of radiolabeled protein from immobilized cells was parallel to that of Folin–Lowry positive material over a range of 0–60 beads (0–60 mg powder). Cycloheximide (0–100 μg/mL) was shown to progressively inhibit the incorporation of L-[U-14C]leucine so that the radioactivity present in the initial HCl–NaCl extract (i.e., [14C]leucine) increased as that in the final NaOH extract (i.e., 14C-labeled protein) decreased. Using this new assay for cell protein, free and immobilized cell cultures were found to exhibit virtually identical kinetics of glucose utilization, growth, and patulin production. In addition to providing a means of comparing the specific productivity of free versus immobilized cell preparations, this assay accurately measures the incorporation of [14C]leucine into cellular protein and could be used as a measure of cell viability.