Boron trifluoride-etherate (BF3), one of the Lewis acids, was found to be an efficient acid in the Edman sequencing method, affording the retention of the N-terminal amino acid configuration at the cyclization/cleavage reaction from peptides. Examples for the sequencing of D-Leu-Gly and L-Pro-L-Leu are described, utilizing 7-N,N-dimethylaminosulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) as a fluorogenic Edman reagent. The peptide labelled with DBD-NCS was cyclized/cleaved with 1% BF3 in dichloroethane containing 0.02% ethanethiol at 50 degrees C for 5 min. The resultant DBD-thiazolinone (TZ)-amino acid was separated on a phenylcarbamoylated cyclodextrin column with fluorometric detection at 524 nm with excitation at 387 nm. DBD-TZ-amino acids thus obtained showed remarkable retention of their configuration of alpha-carbon atoms of amino acids (DBD-TZ-L-Leu; 3%, -D-Pro; 6%). Even after 30 min of the cyclization/cleavage reaction at 50 degrees C, DBD-TZ-amino acids retained their configuration to the same degree. On the other hand, DBD-TZ-amino acid obtained by cyclization/cleavage reaction with TFA at 50 degrees C for 5 min was racemized to a great extent (DBD-TZ-L-Leu; 31%, -D-Pro; 48%). BF3 should be the most recommendable acid at the cyclization/cleavage reaction for amino acid sequence and configuration determination of peptides containing D-amino acid residues.

Amino Acid Sequence↗

PubMedDate not supplied

Detection of free radicals generated from hydrogen peroxide, gallic acid and haemoprotein chemiluminescence system by electron spin resonance spectroscopy.

Low-level chemiluminescence is produced in a hydrogen peroxide (H(2)O(2))/gallic acid/haemoprotein system with single broad peaks around 520 nm, regardless of the biological role of the haemoprotein. The free haem iron systems (haemin and haematin systems) gave a higher photon intensity (1.5 x 10(4) and 2.0 x 10(4) cps) than that of the H(2)O(2)/gallic acid/haematoporphyrin system. These results indicated that haem iron plays a significant role in the photon emission of haemoprotein systems. A free radical with a g value of 2. 0058 was detected through a direct electron spin resonance (ESR) method. The photon intensity of the H(2)O(2)/gallic acid/haemoprotein system decreased in the order: HRP > cytochrome c > myoglobin > haemoglobin, and this corresponded to the decrease in radical intensity. These results indicated that the formation of the free radical with a g value of 2.0058 may be the key step for chemiluminescence in the H(2)O(2)/gallic acid/haemoprotein system. A quartet line similar to DMPO-OH adducts and uncomplexed free radicals (g = 2.0058) was detected using the ESR spin-trapping method in the H(2)O(2)/gallic acid/cytochrome c system.

Electron Spin Resonance Spectroscopy↗