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pH and temperature effects on the molecular conformation of the porcine pancreatic secretory trypsin inhibitor as detected by hydrogen-1 nuclear magnetic resonance

Journal Article · · Biochemistry; (United States)
OSTI ID:5580587
; ;

/sup 1/H NMR spectra of the porcine pancreatic secretory trypsin inhibitor (PSTI) have been recorded vs. pH and temperature. Of the two tyrosines, one titrates with a pK of 1.25, while the resonances from the other are pH insensitive in the investigated range 4.8 less than or equal to pH less than or equal to 12. This is consistent with PSTI having one Tyr solvent exposed (Try-20) and the other buried (Tyr-31). The resonances from the lysyl epsilon-CH/sub 2/ protons titrate with a pK of 10.95. The titration is accompanied by a pronounced line broadening, which starts near pH 8.5. Between pH 11.5 and pH 12 the epsilon-CH/sub 2/ resonances recover their low pH line width. Titration curves for the lysines and Tyr-20 reflect single proton ionization equilibria, suggesting that these residues do not interact among themselves. On the basis of double resonance experiments, combined with analysis of chemical shifts, spin-spin couplngs, and line widths, all methyl resonances are identified and followed as functions of pH and temperature. The ..gamma..-CH/sub 3/ doublet from the N-terminal Thr-1 is assigned by comparison between spectra of forms I and II of the inhibitor, the latter lacking the first four residues of form I. The ..beta..-CH/sub 3/ resonance from Ala-7 is also assigned. Proton resonance parameters of methyl groups are shown to afford useful NMR probes for the characterization of local nonbonded interactions, microenvironments, and mobilities.

Research Organization:
Instituto di Chimica delle Macromolecole del CNR, Milan, Italy
OSTI ID:
5580587
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 21:2; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOLOGICAL FUNCTIONS
DOMESTIC ANIMALS
ENZYME INHIBITORS
ENZYMES
FUNCTIONS
HYDROGEN 1
HYDROGEN ISOTOPES
HYDROLASES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MAMMALS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
PEPTIDE HYDROLASES
PH VALUE
RESONANCE
SERINE PROTEINASES
SPECTRA
STABLE ISOTOPES
SWINE
TEMPERATURE EFFECTS
TRYPSIN
VERTEBRATES