Determination of the disulfide bond pairings in bovine transforming growth factor-alpha.
A rapid method for determining the three disulfide bond pairings in bovine transforming growth factor-alpha (bTGF-alpha) was developed by digesting bTGF-alpha with thermolysin followed by separation of the generated peptides by reversed-phase HPLC. The disulfide-bonded peptides were identified by amino acid sequencing and fast atom bombardment mass spectrometry. The disulfide bond pairings in bTGF-alpha were determined to be homologous to those in the human and mouse TGF-alpha molecules. A species of low bioactivity isolated from the folding/oxidation mixture of chemically synthesized bTGF-alpha was demonstrated to contain two incorrect disulfide bonds. These results indicate that mispairing of disulfide bonds in bTGF-alpha significantly reduces the activity of this molecule.
Violand
B N
BN
Animal Sciences Division, Monsanto Company, St. Louis, MO.
Tou
J S
JS
Vineyard
B D
BD
Siegel
N R
NR
Smith
C E
CE
Pyla
P D
PD
Zobel
J F
JF
Toren
P C
PC
Kolodziej
E W
EW
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D013820:Thermolysin / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D016211:Transforming Growth Factor alpha; D013820:Thermolysin; D004229:Dithiothreitol