Comparison of five procedures for the purification of insoluble elastin.
Elastin is an insoluble, highly cross-linked protein, providing elasticity to organs like lung. aorta, and ligaments. Despite its remarkable mechanical properties. elastin has found little use as a biomaterial. Purification of intact elastin from elastic fibres presents a major challenge, among others for the intimate interwoveness of elastin and microfibrils. Insoluble elastin preparations tend to calcify, which may be due to calcium-binding microfibrillar (e.g. fibrillin). In this study, elastin was purified from horse ligamentum nuchae using five different procedures. One procedure is based on treatment with 0.1 M NaOH, another on autoclaving and treatment with cyanogen bromide. Three other procedures are based on combinations of extraction steps and enzyme digestions. Purity of preparations was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, amino acid analysis, bright field immunofluorescence and transmission electron microscopy. The procedure involving extractions/enzymes combined with an early application of 2-mercaptoethanol and cyanogen bromide gives a highly pure elastin preparation. Electron microscopic analysis showed that this preparation is devoid of microfibrillar components. This procedure is therefore the method of choice for preparation of insoluble elastin as a biomaterial for tissue engineering.
Daamen
W F
WF
Department of Biochemistry, University Medical Centre Nijmegen, Netherlands.
Hafmans
T
T
Veerkamp
J H
JH
Van Kuppevelt
T H
TH
D000096:Acetone / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D017364:Collagenases / Q000494:pharmacology; D003488:Cyanogen Bromide / Q000494:pharmacology; D004549:Elastin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D019791:Guanidine / Q000494:pharmacology; D006736:Horses; D006358:Hot Temperature; D008022:Ligaments / Q000737:chemistry / Q000187:drug effects; D008623:Mercaptoethanol / Q000494:pharmacology; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D012972:Sodium Hydroxide / Q000494:pharmacology; D012995:Solubility; D012997:Solvents / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D012997:Solvents; D000096:Acetone; D012972:Sodium Hydroxide; D008623:Mercaptoethanol; D004549:Elastin; D014357:Trypsin; D017364:Collagenases; D019791:Guanidine; D003488:Cyanogen Bromide