ESI-MS in the study of the activity of α-chymotrypsin in aqueous surfactant media

被引:5
作者
De Angelis, F [1 ]
Di Tullio, A
Del Boccio, P
Reale, S
Savelli, G
Spreti, N
机构
[1] Univ Aquila, Dipartimento Chim Ingn Chim & Mat, I-67100 Laquila, Italy
[2] Univ Perugia, Dipartimento Chim, I-60123 Perugia, Italy
关键词
D O I
10.1039/b302931j
中图分类号
O62 [有机化学];
学科分类号
070303 ; 081704 ;
摘要
The catalytic activity of alpha-chymotrypsin on a model and a peptide substrate, in the supramolecular system "enzyme-surfactant" in water solution, has been studied by electrospray ionization mass spectrometry. Hydrolysis of N-succinyl-L-phenylalanine p-nitroanilide as the model compound, catalysed by alpha-chymotrypsin in the presence of monomeric cetyltributylammonium bromide, has been followed by UV and ESI-MS detection. Kinetic data, which are essentially identical independent of their determination techniques, show a twelve fold improvement of the enzyme catalytic efficiency when compared with the reaction carried out in the absence of the additive. Once validated, the ESI-MS technique was used to study the hydrolytic activity of the enzyme on a peptide substrate like substance P; it is worth emphasising that the spectrophotometric detection cannot be employed on peptides, where the chromophores are untouched by the hydrolytic process. Substance P hydrolyses in aqueous surfactant following dichotomic kinetics, which are initially rapid but then slow down as the reaction progress. The results presented in this paper are expected to extend studies on biocatalysis in aqueous surfactant media to a wide range of substrates, independent of their spectroscopic properties.
引用
收藏
页码:3125 / 3130
页数:6
相关论文
共 30 条
[1]   α-chymotrypsin superactivity in cetyltrialkylammonium bromide-rich media [J].
Alfani, F ;
Cantarella, M ;
Spreti, N ;
Germani, R ;
Savelli, G .
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 2000, 88 (1-3) :1-15
[2]   MICELLAR ENHANCEMENTS OF RATES OF SN2 REACTIONS OF HALIDE-IONS - THE EFFECT OF HEADGROUP SIZE [J].
BACALOGLU, R ;
BUNTON, CA ;
ORTEGA, F .
JOURNAL OF PHYSICAL CHEMISTRY, 1989, 93 (04) :1497-1502
[3]   EFFECT OF IMMISCIBLE ORGANIC-SOLVENTS ON ACTIVITY STABILITY OF NATIVE CHYMOTRYPSIN AND IMMOBILIZED-STABILIZED DERIVATIVES [J].
BLANCO, RM ;
HALLING, PJ ;
BASTIDA, A ;
CUESTA, C ;
GUISAN, JM .
BIOTECHNOLOGY AND BIOENGINEERING, 1992, 39 (01) :75-84
[4]   Monitoring enzyme catalysis with mass spectrometry [J].
Bothner, B ;
Chavez, R ;
Wei, J ;
Strupp, C ;
Phung, Q ;
Schneemann, A ;
Siuzdak, G .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (18) :13455-13459
[5]   PEPTIDE SEQUENCING BY USING A COMBINATION OF PARTIAL ACID-HYDROLYSIS AND FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY [J].
DEANGELIS, F ;
BOTTA, M ;
CECCARELLI, S ;
NICOLETTI, R .
BIOCHEMICAL JOURNAL, 1986, 236 (02) :609-612
[6]  
DEANGELIS F, 1986, BIOCHEM J, V245, P623
[7]   ELECTROSPRAY IONIZATION FOR MASS-SPECTROMETRY OF LARGE BIOMOLECULES [J].
FENN, JB ;
MANN, M ;
MENG, CK ;
WONG, SF ;
WHITEHOUSE, CM .
SCIENCE, 1989, 246 (4926) :64-71
[8]   IMMOBILIZATION STABILIZATION OF ALPHA-CHYMOTRYPSIN BY COVALENT ATTACHMENT TO ALDEHYDE AGAROSE GELS [J].
GUISAN, JM ;
BASTIDA, A ;
CUESTA, C ;
FERNANDEZLAFUENTE, R ;
ROSELL, CM .
BIOTECHNOLOGY AND BIOENGINEERING, 1991, 38 (10) :1144-1152
[9]   Substance P [J].
Harrison, S ;
Geppetti, P .
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, 2001, 33 (06) :555-576
[10]   KINETIC MONITORING OF ENZYMATIC-REACTIONS IN REAL-TIME BY QUANTITATIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY [J].
HSIEH, FYL ;
TONG, X ;
WACHS, T ;
GANEM, B ;
HENION, J .
ANALYTICAL BIOCHEMISTRY, 1995, 229 (01) :20-25