Polypeptide hormone interaction. 3. Conformational changes of glucagon bound to lysolecithin.

The Turner 210 spectrofluorometer was used for fluorescence mea;;urements. The Cary model 15 spectrophotometer was used to obtain absorption and difference spectra. A Cary 60 spectropolarimeter, equipped with a Pockels cell, was used to measure circular dichroism. Solutions had optical densities, in the wave length region of measurement, below 2.0. The data are reported as mean residue ellipticities, [0], in units of degrees square centimeter per dmole: [0] = [lo0 0]/[1 (cm) 1M(molarity)] where 0 is the observed ellipticity. The peptide residue molar&y was calculated as the product of the number of amino acids, less 1, and the peptide molarity. The concentration of the various polypeptides and hormones was determined by absorbance measurements at 280 nm. The molar extinction coefficients at 280 nm were calculated from their tryptophan and tyrosine content using values of 5500 for the former and 1200 for the latter (2). The materials were the same as described in the accompanying paper (1). The carboxy-terminal o&a-, nona-, and decapeptides of glucagon were a gift of Dr. K. Ltibke, Schering AG, West Berlin. Their absorption spectra were characteristic of tryptophan. On thin layer chromatograms, following the procedure of Brewer et al. (3), there were, in addition to one major spot, one or two minor spots for each peptide. All of the spots were tryptophan positive when sprayed with Ehrlich’s reagent. This heterogeneity could arise from pyrrolidone carboxylic acid formation of the NHz-terminal glutamine of the decapeptide, or by partial deamidation of the asparagine residue. The carboxyl-terminal dodecapeptide was prepared by enzymatic digestion of glucagon with trypsin (n-l-tosylamido-2phenylethyl chloromethyl ketone, Worthington). A ratio of peptide to enzyme of 1OO:l was used in 0.1 M trimethylamine acetate buffer, pH 8.2, at 37”. The digestion, as measured by acid production, was complete in 30 to 45 min. The reaction resulted in three peptides, as seen by thin layer chromatography, one of which was positive with Ehrlich’s reagent. Since the other two peptides do not contain tryptophan, it was possible to observe the behavior of the dodecapeptide by its fluorescence without further purification. Solutions of all the fragments were prepared in methanol at 1 mg per ml. The methanol concentration in the lysolecithin solutions was trivial since very small volumes were added. The amino-terminal 21 residue peptide of glucagon was a gift from Dr. M. Rodbell (4). The ribonuclease S peptide was obtained from Sigma Chemical Co. Guanidine hydrochloride (Mann) was “ultrapure” grade. Poly(n-lysine) was purchased from Gallard-Schlesinger Chemical Co.