Estudo das interações entre enzimas e polímeros: efeito do poli(etileno glicol) na atividade e na conformação estrutural de enzimas. Adsorção de enzimas sobre superfícies sólidas / Study on the interactions between enzymes and polymers: Influence of polyethylene glycol on the activity and conformation of enzymes. Adsorption of enzymes onto solid surfaces

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

This work aimed to investigate the interactions between enzymes and polymers in solution and also the adsorption behavior of these enzymes on solid surfaces. For that reason it was divided into two parts. In the first part, the influence of poly(ethylene glycol) (PEG), a polymer considered inert and utilized in several biotechnological processes, on the enzymatic activity and structure of the enzyme was studied by means of UV spectrophotometry, calorimetric titration, circular dichroism (CD) and small angle X-ray scattering (SAXS). Glucose-6-phosphate dehydrogenase (G-6-PDH) and hexokinase (HK) were chosen because of their large application in clinical analysis for determination of glucose in the blood strain. Alcohol dehydrogenase (AD), which is widely used to determine alcohol concentration in various samples, was also used. Quantitative results, in a low enzyme concentration range, indicated a strong influence of PEG on the enzymes activity. The calorimetric measurements revealed no favorable interactions between enzyme and polymer, but indicated favorable interactions between PEG and co-enzyme NADP+. In a higher concentration range, SAXS results showed that PEG also exerts a significant effect on the enzyme aggregation process. This work showed that PEG shall no longer be treated as an inert polymer since it interferes in the enzyme activity and structure. The enzymes are complex macromolecules and PEG interacts differently with each one, deserving special attention in each case. In the second part of the work, the adsorption behavior of creatine phosphokinase (CPK) and hexokinase (HK) onto silicon wafers was studied by means of contact angle measurements, in situ ellipsometry and atomic force microscopy (AFM) in water. CPK was chosen due to its large application on the diagnosis of several muscle disorders. This work revealed that the adsorption mechanism of CPK on silicon surfaces is strongly dependent on pH. At pH 4, 6.8 or 9, CPK adsorbed keeping the same conformation as in solution. pectrophotometric measurements revealed a shift on the optimum pH from 6,8 to 9 upon CPK adsorption. HK adsorbed onto glass beads showed higher activity than HK immobilized on silicon wafers. HK covered glass beads could also be reused three times and for a period of at least three weeks. In the contrary, HK covered silicon wafers could not be reused. For practical purposes, HK covered glass beads showed to be a better “biosensor” than HK covered silicon wafers.

ASSUNTO(S)

hexokinase microscopia de força atômica saxs atomic force microscopy biomolecules creatina fosfoquinase elipsometria biomoléculas saxs adsorption polyethylene glycol hexoquinase ellipsometry creatine phosphokinase polietileno glicol

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