Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

Journal Article · Tue Aug 11 04:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00390a040· OSTI ID:5721039

Tuechsen, E; Hayes, J M; Ramaprasad, S; Copie, V; Woodward, C

Solvent exchange of /sup 18/O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectroscopy. Buried water is labeled by equilibrium of the protein in /sup 18/O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable /sup 18/O label eluting with the protein in 10-300 s is determined by an H/sub 2/O-CO/sub 2/ equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one /sup 18/O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven /sup 18/O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent /sup 18/O labeling of carboxyl groups at low pH. In addition to /sup 18/O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent and hydrogen-bonded main chain O, and their pH/sub min/ is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.

• Research Organization: Univ. of Minnesota, St. Paul
• OSTI ID: 5721039
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:16; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMALS
BARYONS
BODY
CATTLE
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ELEMENTARY PARTICLES
ENDOCRINE GLANDS
ENZYME INHIBITORS
ENZYMES
EVEN-EVEN NUCLEI
FERMIONS
FILTRATION
GLANDS
HADRONS
HEAVY WATER
HYDROGEN COMPOUNDS
HYDROGEN TRANSFER
HYDROLASES
ISOTOPES
ISOTOPIC EXCHANGE
LIGHT NUCLEI
MAMMALS
MASS SPECTROSCOPY
MOLECULAR STRUCTURE
NMR SPECTRA
NUCLEI
NUCLEONS
ORGANS
OXYGEN 18
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PANCREAS
PEPTIDE HYDROLASES
PH VALUE
PROTONS
RUMINANTS
SEPARATION PROCESSES
SERINE PROTEINASES
SPECTRA
SPECTROSCOPY
STABLE ISOTOPES
TRYPSIN
VERTEBRATES
WATER