Uniform sup 13 C isotope labeling of proteins with sodium acetate for NMR studies: Application to human carbonic anhydrase II

Journal Article · Tue May 07 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00232a017· OSTI ID:5392885

Venters, R S; Calderone, T L; Spicer, L D; Fierke, C A ^[1]

---

1. Duke Univ., Durham, NC (USA)

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of ({sup 13}C{sub 6}, 99%)glucose. The authors demonstrate here that uniformly (>95%) {sup 13}C and {sup 15}N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium (1,2-{sup 13}C{sub 2},99%)acetate as the sole carbon source and ({sup 15}N, 99%)ammonium chloride as the sole nitrogen source. In addition, the authors demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium (1-{sup 13}C, 99%)acetate and sodium (2-{sup 13}C, 99%)acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the {sup 1}H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium (1,2-{sup 13}C{sub 2}, 99%)acetate growth media is considerably less than the cost of the ({sup 13}C{sub 6}, 99%)glucose growth media. They report here the first published {sup 13}C and {sup 15}N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.

• OSTI ID: 5392885
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:18; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ACETATES
ALKALI METAL COMPOUNDS
ANIMALS
CARBON 13
CARBON ISOTOPES
CARBON-OXYGEN LYASES
CARBONIC ANHYDRASE
CARBOXYLIC ACID SALTS
DOUBLE LABELLING
ENZYMES
EVEN-ODD NUCLEI
HYDRO-LYASES
ISOTOPES
LABELLING
LIGHT NUCLEI
LYASES
MAGNETIC RESONANCE
MAMMALS
MAN
METALLOPROTEINS
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PRIMATES
PROTEINS
RESONANCE
SODIUM COMPOUNDS
STABLE ISOTOPES
VERTEBRATES