A highly specific and quantitative method for determining type III/I collagen ratios in tissues.
The distribution of type I and type III collagens in rat, bovine and human skin were examined by a quantitative 2-D CNBr peptide mapping method. The procedure involved the solubilization of tissues by digestion with CNBr, radioactive labeling in vitro by [3H]-NaBH4 in dimethylformamide, reduction by mercaptoethanol, a second CNBr digestion and 2-D (isoelectric focusing and NaDodSO4 electrophoresis) mapping. The amounts of type I and type III collagen peptide spots in the fluorographs of 2-D maps were analyzed by 2-D scanning densitometer/analyzer. Mixtures containing various ratios of purified type I and type III collagen were used to obtain a standard curve. Using this procedure we were able to determine that in adult human skin (age range 35-65 years) 22% (+1.3%) of the labelled collagen is type III. This value is significantly higher than that was previously estimated by less accurate methods.
Cheung
D T
DT
Department of Biochemistry, School of Medicine, University of Southern California, Los Angeles.
Benya
P D
PD
Perelman
N
N
DiCesare
P E
PE
Nimni
M E
ME
D000328:Adult; D000818:Animals; D002417:Cattle; D003094:Collagen / Q000032:analysis* / Q000592:standards; D003488:Cyanogen Bromide; D005333:Fetus / Q000737:chemistry; D006801:Humans; D010449:Peptide Mapping / Q000379:methods*; D051381:Rats; D011916:Rats, Inbred F344; D012015:Reference Standards; D012867:Skin / Q000737:chemistry; D014018:Tissue Distribution
D003094:Collagen; D003488:Cyanogen Bromide