The cleavage and adsorption of parathyroid hormone at high dilution: implications for receptor binding studies.
Like other polypeptide hormones, purified intact parathyroid hormone (1-84)parathyroid hormone is notoriously unstable and is subject to large adsorptive losses in routine laboratory manipulation. The present studies were undertaken with 125I-labeled hormone to quantitate the problem and to develop preventative measures, particularly with concentrations of physiological interest, 1 . 10(-10) M. It was found that spontaneous cleavage of the hormone takes place upon its incubation in air or oxygen. This can be prevented by the presence of mercaptoethanol or by plasma levels of cysteine and ascorbate. Under non-cleavage conditions, adsorption was found to be extensive on all materials tested. This adsorption increased with time up to 2 h, was independent of ionic strength, increased with increasing temperature and was presumed to involve hydrophobic interactions. Under given conditions, adsorption was proportional to concentration (constant percentage). However, at very high concentrations, 1 . 10(-6) M, adsorption was markedly reduced. Adsorption was minimized at low pH (2). Bovine serum albumin reduced adsorption under all conditions when present at concentrations of 2 mg/ml or more. Coating laboratory ware with cetyl alcohol also was helpful. Using optimal conditions, cleavage is prevented and losses are less than 5% at neutral pH, and under 2% at pH 2.
The cleavage and adsorption of parathyroid hormone at high dilution: implications for receptor binding studies. Like other polypeptide hormones, purified intact parathyroid hormone (1-84)parathyroid hormone is notoriously unstable and is subject to large adsorptive losses in routine laboratory manipulation. The present studies were undertaken with 125I-labeled hormone to quantitate the problem and to develop preventative measures, particularly with concentrations of physiological interest, 1 . 10(-10) M. It was found that spontaneous cleavage of the hormone takes place upon its incubation in air or oxygen. This can be prevented by the presence of mercaptoethanol or by plasma levels of cysteine and ascorbate. Under non-cleavage conditions, adsorption was found to be extensive on all materials tested. This adsorption increased with time up to 2 h, was independent of ionic strength, increased with increasing temperature and was presumed to involve hydrophobic interactions. Under given conditions, adsorption was proportional to concentration (constant percentage). However, at very high concentrations, 1 . 10(-6) M, adsorption was markedly reduced. Adsorption was minimized at low pH (2). Bovine serum albumin reduced adsorption under all conditions when present at concentrations of 2 mg/ml or more. Coating laboratory ware with cetyl alcohol also was helpful. Using optimal conditions, cleavage is prevented and losses are less than 5% at neutral pH, and under 2% at pH 2.