MA,
BROVETTO-CRUZ,
AND LI
Pituitary Lactogenic Hormone. Reaction of Tetranitromethane with Ovine Hormone* Lin Ma,t Jorge Brovetto-Cruz,$ and Choh Hao Li
All seven tyrosyl residues in ovine prolactin were found to react with tetranitromethane. Complete nitration did not reduce the biological activity of the hormone. However, it did increase the rate at which the protein was digested by trypsin. Spectrophotometric titrations of the native hormone in KC1 solution showed that one of the tyrosyl residues is “buried” within the protein molecule and becomes ionized only after extensive alkali denaturation. The remaining six tyrosines were ionized with a highly ABSTRACT:
T
etranitromethane C(NO& is a specific, mild reagent for the nitration of tyrosyl residues in proteins (Sokolovsky et al., 1966) and has been used for functional and structural studies of proteins (Vallee and Riordan, 1969). However, none of the protein hormones have previously been studied by means of this reagent. In the present work we have investigated the reaction of C(NOs)4 with ovine prolactin, the primary structure of which has recently been elucidated (Li et al., 1969). Details of the study are reported herein.
abnormal apparent pK, and the protein underwent irreversible denaturation at pH values above 11.8. However, when the protein was dissolved in guanidine hydrochloride, the ionization of the tyrosines became normal. In contrast to the behavior of the native hormone, the ionization behavior of the nitrated protein was essentially the same in KCl solution as in guanidine hydrochloride solution. These findings suggest that nitration produced significant conformational changes in the protein molecule without altering the biological properties of the hormone.
For the determination of molar extinction coefficient, the lyophilized monomeric fraction of ovine prolactin was allowed to equilibrate with atmospheric moisture at room temperature for 4 days before nitrogen and spectral determinations were made. Nitrogen content was determined by the micro-Dumas method. Spectra were taken on solutions of accurately weighed samples of the material (5-10 mg) dissolved in 5 or 10 ml of 0.1 N acetic acid. From the spectra taken between 360 and 260 mp on a series of solutions, the absorbancy of the protein at 278 mp was determined. The molar extinction coefficient at 278 mp was calculated, using Experimental Procedure the computed values of 22,600 for the molecular weight Prolactin was isolated from fresh sheep pituitary glands and 16.92% for the nitrogen content (Li et ai., 1969). by the procedure of Li and coworkers (Cole and Li, 1955; The amino acid analysis was carried out according to the Li et al., 1969). Tetranitromethane was obtained from procedure of Spackman et al. (1958) in a Beckman amino Aldrich Chemical Co., Inc., Milwaukee, Wis., and trypsin acid analyzer, Model 120-C. The nitrated protein was hydro(lot no. 63317, treated with L-1-tosylamido-2-phenylethyl lyzed with constant-boiling HC1 in a sealed, evacuated glass chloromethyl ketone) from Calbiochem, Los Angeles, tube for 22 hr at 110’. To effect a clear separation of 3 3 Calif. Guanidine hydrochloride, from Eastman Organic dinitrotyrosine, which under normal operating conditions Chemicals of Rochester, New York, was purified with was eluted with valine, it was necessary to change the starting Norit and recrystallized twice from 95 ethanol. 3-Nitrotime of the second buffer from 60 to 85 min. In order to tyrosine and 3,5-dinitrotyrosine, from K & K Laboratories ascertain whether 3-nitrotyrosine and 3,5-dinitrotyrosine of Hollywood, Calif., were recrystallized from water and were destroyed during acid hydrolysis of the protein samples, dried in a desiccator containing phosphorous pentoxide. known amounts of tyrosine and the two nitro derivatives All other chemicals used were of reagent grade. were subjected to the same hydrolytic conditions, for 22 and All spectra were read on a Beckman DK-2A recording 68 hr. spectrophotometer using an optical path of 1.OO cm. AbsorpThe spectrophotometric titrations were carried out in a tion in the range 340-360 mp was used to correct for light Beckman DK-2A spectrophotometer using the difference scattering, as described by Beavan and Holiday (1952). spectra technique described previously (Bewley et at., 1969; Brovetto-Cruz and Li, 1969). Protein was dissolved in 10-20 ml of either 0.15 M KC1 or 6 M guanidine hydrochloride * From the Hormone Research Laboratory, University of California, at a concentration of approximate~y mg,ml. For the forward San Francisco, California. Receiued January 20, 1970. Paper XXIX in titrations, the SOlutiOns Of native prolactin were adjusted the Pituitary Lactogenic Hormone series. This work was supported to an initial pH of 7.8 and solutions of the nitrated protein by grants from the U. S. Public Health Service, the Allen Foundation, and the Geffen Foundation. were adjusted to a pH of 4.7. Spectra of the native hormone t On leave and a grantee from the Chinese University of Hong I
