Quantitation of elastin in tissues and culture: problems related to the accurate measurement of small amounts of elastin with special emphasis on the rat.
Both rat and sheep elastin can be quantified by measurement of discrete peptides released from the insoluble protein by thermolysin digestion. These peptides are easily visualized and measured by HPLC. With the sheep the tallest peak on the chromatogram represents the VGVPG pentapeptide derived from a repeating sequence seen in elastin from many species. This repeating sequence allows for amplification of the signal significantly above background so that accurate quantitation can be carried out. The measurement is reproducible over a wide range of protein concentrations. With the rat however the pentapeptide is not present but appears to be replaced by other repeating sequences. We quantitated and determined amino acid sequence on 8 peaks present in the early portion of the chromatogram for purposes of quantifying rat elastin. That signal most reliably present over a range of concentrations was tyrosyl-glycine (YG) which eluted at 8.5 minutes. We have used YG as a basis for quantitation of rat elastin both from tissues and tissue culture. We have also shown that the desmosine crosslinks are not constant in elastin produced in a neonatal rat smooth muscle culture system but vary with the age of the culture. We thus propose that an index of maturation be considered for a given elastin in the form of mumoles of crosslink per gram of elastin so as to better define its quality.
Sandberg
L B
LB
Connective Tissue Laboratory, Pettis Memorial Veterans' Hospital, Loma Linda, CA 92357.
Roos
P J
PJ
Pollman
M J
MJ
Hodgkin
D D
DD
Blankenship
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D046508:Culture Techniques; D003895:Desmosine / Q000378:metabolism; D004549:Elastin / Q000032:analysis* / Q000737:chemistry; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats / Q000378:metabolism*; D011919:Rats, Inbred Strains; D012756:Sheep; D013820:Thermolysin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D003895:Desmosine; D004549:Elastin; D013820:Thermolysin