Capillary electrophoresis of the proteolytic activity of the stratum corneum obtained by tape stripping.
Serine proteases and some cathepsins are present in the stratum corneum. They are known to play a significant role in the pathophysiological mechanism of several dermatological conditions (e.g. atopic dermatitis) and in the induction of itch. Tape stripping of skin is a simple technique used in the investigation of skin barrier function and in the penetration of topically applied drugs. Herein, we show that CE, under stacking conditions, is a well-suited technique to measure the proteolytic activity of enzymes in the stratum corneum. Disks of about 6 mm (id) were cut from adhered tapes and submerged directly in a buffer containing the appropriate peptide substrate. After incubation, the split peptides were separated and detected directly by CE at 214 nm in a borate buffer. The esterase activity on N-benzoyl-tyrosine ethyl ester and the amidase activity on succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and the splitting of hemoglobin were detected by CE. The esterase activity was the highest when compared to the proteolytic activities. Skin scratching increased the enzymatic activity adhered to the tapes. The CE offered over the traditional end-point colorimetric methods the ability to measure the low enzymatic activity and the ability to detect the released peptides directly. This technique is simple, non-invasive, easy to perform and uses non-expensive substrates. It can be useful in quantifying cathepsins and serine proteases in the skin.
Department of Dermatology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Alexandru D P
AD
Papoiu
Gil
G
Yosipovitch
Zak
Z
Shihabi
D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods*; D004817:Epidermis / Q000201:enzymology*; D006801:Humans; D006867:Hydrolases / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D059748:Proteolysis; D013048:Specimen Handling / Q000379:methods*
D006867:Hydrolases