摘要:

The interactions of [1‐hemi‐(3‐13C)cystine]oxytocin and [1‐hemi‐D‐(3‐13C) cystine]oxytocin with bovine neurophysin‐II have been studied in aqueous solution under various conditions of pH using carbon‐13 (13C) nuclear magnetic resonance spectroscopy (n.m.r.). We have monitored changes in13C chemical shifts (δ), spin‐lattice relaxation times (T1) and linewidths (πT2*)‐1of the [1‐hemi‐(3‐13C)cystine] oxytocin as a function of the concentration of added neurophysin. [1‐Hemi‐d‐(3‐13C)cystine]oxytocin does not interact with neurophysin and shows no changes in the above spectral parameters as a function of increasing protein concentrations. The13C chemical shifts have been monitored as a function of pH for both13C‐enriched, diastereoisomeric oxytocin analogs in the presence and absence of neurophysin. The midpoints of the pH‐titration curves for [1‐hemi‐(3‐13C)cystine]oxytocin and [1‐hemi‐d‐(3‐13C) cystine]oxytocin in the absence or presence of neurophysin are 6.2 and 5.5, respectively. T1values are independent of pH for both peptides in free solution, and the rotational correlation times (tc) obtained for the peptides are of the order of magnitude expected from the Stokes‐Einstein relation (5.0 times 10‐10sec rad‐1). T1values decrease and linewidths increase for [1‐hemi‐(3‐13C)cystine]oxytocin in the presence of neurophysin. Fast‐exchange on the n.m.r. time scale occurs at low pH. Only broad lines can be detected at neutral pH rendering13C T1measurements difficult. It appears that increasing the concentration of neurophysin in the presence of [1‐hemi‐(3‐13C)cystine]oxytocin leads to aggregation of the system as judged by the13C T1results and corresponding rotational correlation times. In light of the significant changes in T1values and small changes in δ values, it would seem that T1measurements are more accurate monitors of peptide‐protein interactions than are13C chemical shifts when the interaction of the peptide with the protein doe

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1979.tb01852.x

出版商:Blackwell Publishing Ltd    

年代:1979

数据来源: WILEY

摘要:

Phenylalanine chloromethyl ketone covalently attached to porous glass beads was synthesized to serve as a solid‐phase active site directed inhibitor of chymotrypsin‐like proteolytic enzymes. The solid‐phase reagent inhibited 23 nmol of bovine chymotrypsin per gram of glass and covalently bound 30 nmol of protein per gram of glass. Sepharose‐bound lysine chloromethyl ketones were synthesized to serve as inhibitors of trypsin‐like enzymes. Sepharose‐MethionylLysyl chloromethyl ketone inactivated and bound about 6.5 nmol of enzyme per ml of settled gel. In a preliminary experiment, a cyanogen bromide cleavage of the methionine residues showed that it should be possible to release all peptides but the peptide containing the active‐site histidine. The immobilized trypsin was also reduced, carboxymethylated and digested with chymotrypsin. The potential of the solid‐phase approach is in the isolation of a specific serine proteinase and in the sequence determination of residues surrounding the active

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1979.tb01853.x

出版商:Blackwell Publishing Ltd    

年代:1979

数据来源: WILEY

摘要:

The 1‐dimethylamino‐5‐naphthalene sulfonyl (dansyl) derivative of alanyllsyl chloromethane was synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active site of trypsin via affinity labelling. The potential of dansylalanyllysyl chloromethane lies in its high degree of selectivity and markedly faster rate of enzyme inactivation when compared to previously synthesized, single residue affinity label chromophores. This permits the practical utilization of stoichiometric amounts of the inhibitor to achieve 100% inactivation of trypsin, even at high dilutions.The transfer of energy between the four tryptophan residues of trypsin and the bound dansyl group has been investigated in the fluorescent inhibitor‐enzyme conjugate. From transfer efficiency measurements mean distances of 19.0 Å and 19.3 Å between the point of attachment of the dansyl group and the four tryptophan residues of trypsin have been calculated. These compare well with the mean value of 18.8 Å derived from calculations based on crystallo‐graphic m

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1979.tb01854.x

出版商:Blackwell Publishing Ltd    

年代:1979

数据来源: WILEY

摘要:

A rapid alternative method is presented for the determination of pyridoxal 5′‐phosphate (pyridoxal‐P). The method involves the colorimetric analysis of thiocyanate liberated from S‐cyanohomocysteine (Hcy(CN)) in the presence of cyanide when catalyzed by the pyridoxal‐P dependent enzyme, γ‐cyano‐α‐aminobutyric acid (γ‐CNabu)‐synthase (Hcy(CN) thiocyano‐lyase [adding CN]). The rate of formation of thiocyanate is determined by the increase in absorbance at 470nm on treatment of the enzymatic rea

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1979.tb01855.x

出版商:Blackwell Publishing Ltd    

年代:1979

数据来源: WILEY

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1979.tb01856.x

出版商:Blackwell Publishing Ltd    

年代:1979

数据来源: WILEY