Submitted to: Journal of American Leather Chemists Association
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 16, 2008
Publication Date: October 1, 2008
Citation: Ramos, M., Latona, R.J., Fortis, L.L., Marmer, W.N. 2008. Identification of Decorin and other Proteins in Bovine Hide during its Processing into Leather. Journal of American Leather Chemists Association. 103(10):324-329. Interpretive Summary: Several hide constituents, such as decorin, are removed, altered or diminished when hides are subjected to tanning treatments to obtain the beautiful, durable and useful material called leather. We had shown that further removal of decorin actually improved the strength, softness, and stretchability of leather. For tanners to intentionally remove more decorin, they need a better way of monitoring the removal and changes of these constituents. Thus, developing a more accurate analytical procedure to measure the concentration of these materials in every step of tanning the hides to leather is important. A newer technique developed and adapted in the present work is first separating the proteins extracted from the hides using SDS-PAGE and then quantifying the deep purple decorin band(s) using the immunochemical technique called Western blotting. We attained improved sensitivity by simultaneously using two different antibodies specific for decorin rather than one. Western blotting has confirmed that earlier-detected species are indeed decorin. Other proteins were identified by using the technique called Matrix Assisted Laser Desorption /Ionization- Time of Flight (MALDI-TOF) mass spectrometry or MALDI-TOF. The fundamental information from this work will guide tanners in processing hides efficiently to high quality leather.
Technical Abstract: The present work attempts to develop a more accurate and reliable assay for decorin concentration in hides as they are processed to leather. The ELISA technique previously developed for decorin analysis is further improved and optimized by treating the samples with chondroitinase-ABC after dialyzing them in the presence of collagenase. Some newer techniques developed and adapted include SDS-PAGE and Western blotting using immuno-colorimetric staining. Probing the blotted protein with a combination of two different antibodies specific for decorin, i.e., PK1 and 6D6, is more specific and sensitive than using either antibody individually. Western Blotting can be used visually and quantitatively to confirm that ELISA-detected species are indeed decorin. The intact proteoglycan as well as the core protein and resulting fragmentations of decorin are determined after treating the samples with collagenase and chondroitinase ABC alone or a combination of these two enzymes. The fate of the different proteins in raw hides to leather is followed by analyzing the separated protein bands in SDS-PAGE gel using MALDI-TOF peptide mapping.