A method for the determination of elastolytic activity is described. The method employs soluble elastin radiolabeled with either lz51 or 3H coupled to Sepharose beads. The assay can detect as little as 1 rig/ml of elastase.
Several different techniques have been employed for the quantitation of elastase. In general, these methods measure either the release of radioactive, fluorescent, or pigmented peptides from insoluble elastin or the increase in ninhydrin-positive TCA-soluble material from soluble elastin (l-9). Each technique contains inherent restrictions such as high background, limited sensitivity, or difficulty in quantitation.
In this paper, we describe an assay for elastase that combines the convenience of a soluble assay with the sensitivity of a radioactive assay. The technique uses 3H-or lz51-labeled soluble elastin coupled to Sepharose beads. Under standard conditions, as little as 1 ng of elastase can be detected by this procedure. The assay has been employed to screen a variety of normal and malignant cells for elastolytic activity.
Methods
Soluble elastin was prepared as described by Keller and Mandl (6) and was tritiated with NaB3H, after incubation with pyridoxal phosphate, as described by Rifkin et al. ( 10) with the following modifications. The elastinoxalic acid solution was first dialyzed overnight in the cold against 3 x 2 liter of glass-distilled water. The pH of the elastin solution was adjusted to pH 8.0 by the addition of one-tenth of a volume of IM sodium phosphate (pH 8.0). To 2 ml of elastin solution (15 mg), 2.48 mgof pyridoxal phosphate (Calbiochem) was added to bring the final concentration of pyridoxal phosphate to 5 mM. The solution was incubated at 37ยฐC for 30 min in the dark, 100 mCi of NaB3H4 (Amersham, 7.1 Ci/mmol) dissolved in 0.1 ml of 0.01 M NaOH was added, and the reaction mixture was then incubated at 20ยฐC for 30 min in a fume hood. At the end of this period, solid pyridoxal phosphate was added until a yellow color persisted. The solution was dialyzed overnight at 5ยฐC against 2 x 2 liter of 0.1 M sodium phosphate (pH 8.0). The [3H]elastin was further purified by gel chromatography on a