Elastin covalently labeled with rhodamine-B-isothiocyanate has been found to be a sensitive substrate for analysis of elastolytic activity in comparison to elastin-orcein and elastin-Congo red. Using elastin-rhodamine in a 2@min test, elastase concentrations as low as 0.1 pg/ml are accurately assayed. Variations in analytical parameters such as temperature, pH, and particle size of the substrate greatly influence the rate of elastolysis. Methods for the determination of elastolytic activity of pancreatic elastase most often rely upon solid elastin labeled with a chromophore or a fluorophore (1). Some of these methods contain analytical drawbacks. Ionic bonded labels, such as orcein and Congo red, are readily eluted from elastin during incubation by nonspecific protein resulting in erroneous elastolytic response (1,2). Additionally, these substrates lack the sensitivity necessary to quantitate minute amounts of elastase unless the period of incubation is excessively long. Within the current methods, certain properties such as pH and temperature of the incubation medium and particle size of the substrate remain unexplored.
We describe in this communication a method for the quantitative analysis of elastolytic activity using as substrate elastin covalently labeled with rhodamine-B-isothiocyanate.
The method is rapid, sensitive, and reproducible, and the label being covalently linked with elastin strongly resists elution by nonspecific protein. Also described are kinetic properties of the enzyme-substrate complex, conditions for assay, and advantages in comparison with elastin labeled with orcein and Congo red.
Procedures based upon similar concepts were first developed by Rinderknecht et al. (1) in which elastin was labeled with fluorescein isothiocyanate or remazol brilliant blue. We selected rhodamine-B-isothiocyanate as label due to its high fluorescent intensity (3), ease of synthesis, and readability of the soluble colored products of elastolysis either in a spectrophotometer or fluorometer.