A Density Labelling Method for the Quantitation of Radioactive Label Recycling in Studies on Individual Protein Turnover

Recycling of radioactive label from degraded protein is a majordrawback in pulse-chase experiments for the measurement of proteinturnover, because it leads to underestimations of degradationconstants. This paper describes a density labelling method forthe quantitation of label recycling into individual proteins,whereby underestimated degradation constants can be corrected.The method requires a density difference between native (light)and density-labelled (heavy) protein of less than 10 g dm^–3and, therefore, allows using ²H₂O concentrations of only 50%(v/v) or even considerably lower. Plants are pulse-labelledwith ^l4C and grown on ²H₂O either during the pulse or followingthe chase. Distribution of ¹⁴C-label between light and heavyprotein is determined from a ¹⁴C-superimposition curve obtainedby isopycnic centrifugation of the purified protein in a CsClgradient. The superimposition curve mathematically is describedby the sum of two single Gaussian curves. It proved impossibleto determine all six unknown parameters of this function byan algorithm. Therefore, mean values (i.e. the peak positionsin the gradient) of light and heavy protein are determined frominternal markers (³H-labelled light or heavy protein). Evaluationof data is performed by a computerized curve fitting programwhich provides a numerical and a graphical result of label recycling.The detection limit of the method is 10% of the minor componentin a heterogenous population of light and heavy protein differingin density by only 8.0 g dm^–3. Key words: Density labelling, deuterium oxide, label recycling, protein degradation, protein turnover