A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples.
Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking in the biosynthesis of normal mature functional insoluble extracellular matrices. In addition, lysyl oxidase has been identified as a possible tumor suppressor. Lysyl oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. In addition, a less sensitive fluorometric method exists that employs nonpeptidyl amine lysyl oxidase substrates and measures hydrogen peroxide production with horseradish peroxidase coupled to homovanillate oxidation. The present study describes a more sensitive fluorescent assay for lysyl oxidase activity that utilizes 1,5-diaminopentane as substrate, and released hydrogen peroxide is detected using Amplex red in horseradish peroxidase-coupled reactions. This method allows the detection of 40 ng of enzyme per 2 ml assay at 37 degrees C and is 7.5 times more sensitive than the currently available fluorometric assay for enzyme activity. This method eliminates the interference that occurs in some biological samples and can be successfully used to detect lysyl oxidase activity in cell culture experiments.
Palamakumbura
Amitha H
AH
Division of Oral Biology, Boston University Goldman School of Dental Medicine, 100 East Newton Street, Boston, Massachusetts 02118, USA.
Trackman
Philip C
PC
D016475:3T3 Cells; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002417:Cattle; D002478:Cells, Cultured; D002863:Chromogenic Compounds / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006719:Homovanillic Acid / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010078:Oxazines / Q000378:metabolism; D008249:Protein-Lysine 6-Oxidase / Q000032:analysis* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014316:Tritium / Q000378:metabolism
D002863:Chromogenic Compounds; C105363:N-acetyl-3,7-dihydroxyphenoxazine; D010078:Oxazines; D014316:Tritium; D006861:Hydrogen Peroxide; D008249:Protein-Lysine 6-Oxidase; D006719:Homovanillic Acid