Isolation and characterization of insoluble collagen of dog hearts.
A procedure for isolating insoluble heart collagen has been developed. The method involves the use of defined optimal conditions of sonication that yield no thermal denaturation of the triple-helical structure nor disruption of the primary structure of the collagen molecules; this is followed by extraction of isolates with nondenaturing agents. The amino acid residues of the isolates are then reacted with dansyl chloride to allow determination of amino-terminal residues and quantification of the collagen. The method has several advantages over existing procedures: (i) There is no other method available for isolation of undenatured insoluble heart collagen in almost pure form (consists of 96% of type I collagen) and in a good yield. Sonication of tissue at or below 4 degrees C for a total of 120 s (15 s sonication repeated 8 times at 120-s intervals) yielded insoluble collagen fibers with 90% yield and a 20-fold purification as determined by the increase in Hyp content of the isolates. Extraction of these isolates with 0.6 M KCl and 1 M NaCl at 4 degrees C resulted in a 22-fold purification with 70% yield, while the classical extraction method with nondenaturing reagents yielded only 5-fold purification. (ii) There has been little study of the derivatization of an insoluble protein (collagen) with dansyl chloride. The Lys residues of collagen could be recovered as epsilon-Dns-Lys in 84% yield from a reverse-phase C-18 column by high-performance liquid chromatography. This assay allows measurement of 0.1-100 nmol epsilon-Dns-Lys. (iii) The method generates direct information concerning the quantity of collagen and its nature with respect to amino groups.
Takahashi
S
S
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, New York 10461.
Zhao
M
M
Eng
C
C
D000818:Animals; D003094:Collagen / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D003619:Dansyl Compounds; D004285:Dogs; D008297:Male; D008855:Microscopy, Electron, Scanning; D009206:Myocardium / Q000737:chemistry*; D011487:Protein Conformation; D012995:Solubility; D013010:Sonication
D003619:Dansyl Compounds; D003094:Collagen; C029038:dansyl chloride