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Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments

1975; Elsevier BV; Volume: 250; Issue: 16 Linguagem: Inglês

10.1016/s0021-9258(19)41077-6

1083-351X

Eric Oldfield, RS Norton, Adam Allerhand,

Protein Structure and Dynamics

Horse heart cytochrome c was purchased from Sigma Chemical Co., St. Louis, MO. (type III and VI) and from Calbiochem, La Jolla, Calif.(A grade).Candida krusei cytochrome c (type VII), hen egg white lysozyme (grade I), horse skeletal muscle myoglobin, sperm whale skeletal muscle myoglobin, Gly-His-Gly, Gly-Phe amide acetate, Gly-Tyr amide hydrochloride, r.-arginine hydrochloride, N-bromosuccinimide, and gadolinium oxide (99.9%) were purchased from Sigma Chemical Co.L-Tryptophan was obtained from Matheson, Coleman & Bell, Norwood, Ohio.Oxindole was purchased from Aldrich Chemical Co., Milwaukee, Wis.Lanthanum oxide (ultrapure grade) was obtained from Alfa Products, Beverly, Mass.'X0 (28.5's) '"C) was purchased from Monsanto Research Corp., Miamisburg, Ohio.K%N (85 to 90% I%) was obtained from Mallinckrodt Chemical Works, St. Louis, MO.Unless otherwise stated, protein solutions were concentrated in a model 52 or 402 stirred ultrafiltration cell (Amicon Corp., Lexington, Mass.) equipped with a suitable Diaflo membrane.Samples of ferrocytochrome c and ferricytochrome c in H,O were prepared as described previously (2).Samples of cyanoferricytochrome c were prepared by addition of KCN in phosphate buffer to a buffered solution of ferricytochrome c.Solutions of horse heart f'erri-and ferrocytochrome c in D,O were prepared by concentrating and diluting 4 g of protein five times over a &hour period, each time using 50 ml of a 0.05 M phosphate/O.1 M NaCl solution in D,O (pH meter reading 6.7), at 25".A solution of horse heart ferrocytochrome c in D,O was prepared by adding solid sodium dithionite to a D,O solution of ferricytochrome c under argon.Hen egg white lysozyme was purified on a column of DEAE-Sephadex.' Samples in H,O were prepared by dilution of a 20 mM stock solution of protein.pH was adjusted with 5 M HCl or NaOH.A 'R. S.