Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes.
Peptides purified by HPLC are often in the form of a trifluoroacetate (TFA) salt, because trifluoroacetic acid is used as a solvent in reversed-phase HPLC separation. However, the potential effects of this contaminant in culture systems have not been addressed previously. TFA (10(-8) to 10(-7) M) reduced cell numbers and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and neonatal mouse calvariae, indicating that the effect is not specific to one cell type or to one species of origin. When the activities of the TFA and hydrochloride salts of amylin, amylin-(1-8), and calcitonin were compared in osteoblasts, cell proliferation was consistently less with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above 10(-9) M in whatever cell or tissue type. Such peptides should be converted to a hydrochloride or biologically equivalent salt before assessment of their biological effects is undertaken.
Cornish
J
J
Department of Medicine, University of Auckland, Auckland 1001, New Zealand. j.cornish@auckland.ac.nz
Callon
K E
KE
Lin
C Q
CQ
Xiao
C L
CL
Mulvey
T B
TB
Cooper
G J
GJ
Reid
I R
IR
D000682:Amyloid / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D000838:Anions / Q000302:isolation & purification / Q000494:pharmacology; D002116:Calcitonin / Q000302:isolation & purification / Q000494:pharmacology; D015740:Calcitonin Gene-Related Peptide / Q000302:isolation & purification* / Q000494:pharmacology; D002358:Cartilage, Articular / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards*; D002455:Cell Division / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology*; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005333:Fetus / Q000166:cytology; D006851:Hydrochloric Acid; D058228:Islet Amyloid Polypeptide; D051379:Mice; D009924:Organ Culture Techniques / Q000379:methods / Q000592:standards; D010006:Osteoblasts / Q000166:cytology*; D051381:Rats; D012886:Skull / Q000166:cytology; D014269:Trifluoroacetic Acid / Q000494:pharmacology*
D000682:Amyloid; D000838:Anions; D058228:Islet Amyloid Polypeptide; D002116:Calcitonin; D014269:Trifluoroacetic Acid; D015740:Calcitonin Gene-Related Peptide; D006851:Hydrochloric Acid