Abstract
Abstract The structure of porcine thyrocalcitonin has been evaluated by circular dichroism, optical rotatory dispersion, infrared spectroscopy, and fluorescence. The degree of helical structure was estimated by circular dichroism (222 mµ) and optical rotatory dispersion (233 mµ) at the n → π* transition of the α helix. The optical activity at this transition suggests that thyrocalcitonin contains approximately 10% α-helical structure in aqueous solution. The spectrum of thyrocalcitonin in 6 m guanidine was similar to that of a randomly coiled polypeptide. In 2-chloroethanol, thyrocalcitonin formed a structure containing approximately 50% α helix. The near ultraviolet circular dichroic spectrum of thyrocalcitonin revealed a major band at 288 mµ, indicating that the tryptophanyl chromophore had restricted rotational freedom. Reduction and alkylation of the amino-terminal heptapeptide ring of thyrocalcitonin produced no significant change in the circular dichroic or optical rotatory spectra. In addition, the effects of alkaline pH and temperature on tryptophanyl emission were similar to the unmodified hormone. These findings preclude a high degree of organized structure in the heptapeptide ring, which is in marked contrast to the amino-terminal hexapeptide ring of oxytocin. These studies indicate that porcine thyrocalcitonin exists predominately in a random coil in aqueous solution. The polypeptide, however, does not appear to possess a rigid conformation and a coil ⇌ helix equilibrium may exist in water with guanidine shifting the equilibrium in favor of the coil and 2-chloroethanol in favor of the helix. The latter type of transition may occur at the receptor site of the hormone in lipid layers or cellular membranes where the water concentration is significantly reduced.
Highlights
The major dichroic activity is centered near 200 rnp ([0’12, = -9,700) the region of the ellipticity band of the peptide bond in an unorganized polypeptide [14]
There is a second, weaker dichroic band near 222 rnF ([e’], = -3,300), the wave length of the n --f r* transition of the Q!helix, which forms a shoulder on the stronger band
In 6 M guanidine the shoulder disappeared and the ellipticit,y at 222 rnp decreased to -1,100, while the major band appears to maintain its rotational strength (Fig. 1)
Summary
TC was isolated in a homogeneous form as previously reported [8]. Biological activity was 200 MRC units per mg as measured by rat bioassay [9]. Protein concentrations were determined by absorbance measurements at 280 rnp 0.1 M acetic acid = 2.1) or by amino acid analysis. Reduction of TC followed by alkylation with iodoacetic acid was performed as outlined previously [8]. Cary model 60 spectropolarimeter equipped with a Pockels cell was used. The optical density of all solutions was close to 2 at 280 mp. Determinations were made in l-cm cells in the near ultraviolet region, and with 0.5-
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