The Primary Structure of Bovine Thyrotropin

Primary Structure of Bovine Thyrotropin.I Vol.246, No. 4each sample contained two or three of the hormonally active alanine by the number of their residues per mole, taken from the components (15).sequence data (10).MuteKuZsCyanogen bromide was obtained from Eastman; phenylisothiocyanate from J. T. Baker Chemical Company, the reagent was redistilled at reduced pressure and stored under nitrogen.Pyridine was redistilled over phthalic anhydride and stored under nitrogen in the cold.Trypsin was the most highly purified preparation supplied by Worthington and was treated with L-(l-tosylamido-2-phenyl)ethyl chloromethyl ketone to inactivate any residual chymotrypsin (16).Dansyl chloride was supplied by Sigma.Amino Acid and Carbohydrate Analyses---Amino acid analyses were made on a Spinco model 120 amino acid analyzer modified for accelerated analysis.Neutral sugars were determined by gas-liquid chromatography and the hexosamines with the amino acid analyzer as previously described ( 17).Samples for amino acid analysis were hydrolyzed, usually for 24 hours, in 6 N HCl ( 14).Variable and sometimes complete destruction of Scarboxymethylcysteine occurred &s well as some loss of tyrosine and methionine particularly when peptides had been eluted from paper, so 50 ~1 of 5% aqueous phenol was added to the hydrolysis mixture and in later stages of the work 5 ~1 of mercaptoacetic acid (18).The latter addition greatly improved recoveries of S-carboxymethylcysteine and methionine.Amino acid analyses of reduced S-carboxymethyl TSH, TSH-ar, and TSH-& carried out under these conditions showed, in contrast to previous determinations (2, 12, 14), a higher number of these residues in agreement with the sequence data.The amino acid analyses were, in the case of the peptides, calculated with 1 stable residue taken as the residue value or the values for several residues were averaged.For reduced S-carboxymethyl TSH and the separate