Human dermal fibroblasts are used as a source of the elastase enzyme.  After lysing the fibroblasts, portions of the lysate containing the elastase enzyme are briefly incubated with test materials followed by the addition of a synthetic elastase substrate, Suc-(Ala₃)-p-Nitroaniline.  Elastase will act upon this substrate to release p-nitroaniline, which can be detected with a spectrophotometer by measuring the absorbance at a wavelength of 405 nm.  An inhibition of the elastase enzyme is denoted by a decrease in the amount of released p-nitroaniline when compared to uninhibited enzyme.

 

MMP-1 Assay

Matrix Metalloproteinase-1 (MMP-1) is a zinc and calcium dependent endopeptidase that is produced and released from both dermal fibroblasts and keratinocytes and functions to break down collagens located in the extracellular matrix.  Both active and inactive MMP-1 released into the culture media can be assayed using a fluorescence-based ELISA.   Briefly, antibodies covalently linked to a solid support will bind any MMP-1 (both active and inactive MMP-1) present in spent culture media samples.  If desired, inactive MMP-1 can be activated at this point by adding p-aminophenylmercuric acetate to the sample.  To quantify the active MMP-1, a fluorogenic substrate linked to a quencher molecule is added and any active MMP-1 present will cleave the peptide linkage between the fluorophore and the quencher molecule.  This cleavage will eliminate the ability of the quencher molecule to inhibit the fluorescent signal of the fluorophore allowing a fluorescent signal that will be proportional to the amount of active MMP-1 present.