Enhancing the peroxidase-like activity of ficin by rational blocking thiol groups for colorimetric detection of biothiols.
Talanta
; 204: 833-839, 2019 Nov 01.
Article
em En
| MEDLINE
| ID: mdl-31357372
ABSTRACT
The peroxidase-like activity of ficin is relatively low, which limits its application. It was found that thiol groups of ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N-ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of ficin, ficin-NEM (ficin-N) and ficin-TCEP-NEM (ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of ficin-N and ficin-TN toward the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by H2O2 was about 2.5-fold and 5-fold increase compared with ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of ficin-N (Kmâ¯=â¯0.31) and ficin-TN (Kmâ¯=â¯0.39) to H2O2 compared with ficin (Kmâ¯=â¯0.58), and ficin-TN had the highest Kcat which increased by 6.5 times and 4.5 times for TMB and H2O2, respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of ficin. Comparing with ficin and ficin-N, ficin-TN had the widest detection range (0.01-16⯵M) and the lowest detection limit (3â¯nM). The practical applications of ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and ficin-TN are promising to apply to the bioanalysis.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peroxidases
/
Ficina
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Cisteína
/
Glutationa
/
Homocisteína
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article