Chymotrypsin adsorption on montmorillonite: Enzymatic activity and kinetic FTIR structural analysis

被引:88
作者
Baron, MH
Revault, M
Servagent-Noinville, S
Abadie, J
Quiquampoix, H
机构
[1] Univ Paris 06, Lab Dynam Interact & React, CNRS, F-94320 Thiais, France
[2] ENSAM, INRA, UFR Sci Sol, F-34060 Montpellier, France
关键词
alpha-chymotrypsin; protein adsorption; enzymatic activity; montmorillonite; Fourier transform infrared spectroscopy;
D O I
10.1006/jcis.1999.6189
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
Soils have a large solid surface area and high adsorptive capacities, To determine if structural and solvation changes induced by adsorption on clays are related to changes in enzyme activity, alpha-chymotrypsin adsorbed on a phyllosilicate with an electronegative surface (montmorillonite) has been studied by transmission FTIR spectroscopy. A comparison of the pH-dependent structural changes for the solution and adsorbed states probes the electrostatic origin of the adsorption, In the pD range 4.5-10, adsorption only perturbs some peripheral domains of the protein compared to the solution. Secondary structure unfolding affects about 15-20 peptide units. Parts of these domains become hydrated and others entail some self-association. However, the inactivation of the catalytic activity of the adsorbed enzyme in the 5-7 pD range is due less to these structural changes than to steric hindrance when three essential imino/amino functions, located close to the entrance of the catalytic cavity (His-40 and -57 residues and Ala-149 end chain residue), are oriented toward the negatively charged mineral surface, When these functions lose their positive charge, the orientation of the adsorbed enzyme is changed and an activity similar to that in solution at equivalent pH is recovered. This result is of fundamental interest in all fields of research where enzymatic activity is monitored using reversible adsorption procedures. (C) 1999 Academic Press.
引用
收藏
页码:319 / 332
页数:14
相关论文
共 67 条
[1]   ADSORPTION OF LYSOZYME AND OVALBUMIN BY CLAY - EFFECT OF CLAY SUSPENSION PH AND CLAY MINERAL TYPE [J].
ALBERT, JT ;
HARTER, RD .
SOIL SCIENCE, 1973, 115 (02) :130-136
[2]  
ALIEV VS, 1976, VESTN MOSK U BIOL PO, V31, P67
[3]  
ARMSTRONG DAVID E., 1964, SOIL SCI, V98, P39, DOI 10.1097/00010694-196407000-00007
[4]   QUANTITATIVE STUDIES OF THE STRUCTURE OF PROTEINS IN SOLUTION BY FOURIER-TRANSFORM INFRARED-SPECTROSCOPY [J].
ARRONDO, JLR ;
MUGA, A ;
CASTRESANA, J ;
GONI, FM .
PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, 1993, 59 (01) :23-56
[5]  
BARON D, 1991, LOGICIELS CHIMIE, P282
[6]  
BARON MH, 1993, FIFTH INTERNATIONAL CONFERENCE ON THE SPECTROSCOPY OF BIOLOGICAL MOLECULES, P109
[7]   PRERESONANCE RAMAN-SPECTRA OF TYROSINE AND IODOTYROSINE AT PH1 AND 11,5 [J].
BARON, MH ;
DELOZE, C ;
MEJEAN, T ;
COULANGE, MJ .
JOURNAL DE CHIMIE PHYSIQUE ET DE PHYSICO-CHIMIE BIOLOGIQUE, 1983, 80 (10) :729-737
[8]  
BARON MH, 1972, BIOPOLYMERS, V11, P2063
[9]  
BIRKTOFT JJ, 1975, PDB 2CHA
[10]   STRUCTURE AND MECHANISM OF CHYMOTRYPSIN [J].
BLOW, DM .
ACCOUNTS OF CHEMICAL RESEARCH, 1976, 9 (04) :145-152