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 enzymes was studied by means of UV spectrophotometry, calorimetric titration, circular dichroism (CD)

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... d 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. Spectrophotometric 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. Parte 2: Adsorção de enzimas sobre superfícies sólidas Figura 1: Espectro de absorção de Reagente de Bradford (CCBG) na presença (curva azul) e na ausência de proteína (curva vermelha). (página 57) Figura 2: Medida de absorbância a 595 nm de soluções de HK ligada ao Reagente de Bradford (CCBG). (página 58) Figura 3: Cinética de adsorção in situ de CPK (c = 0,005 g.L -1 ) sobre placa de silício a 22,5 ± 0,5 o C e pH 6,8. (página 63) Figura 4. Imagens de topografia dos filmes de CPK e das fronteiras filme-substrato obtidas após remoção do parafilme que cobria metada da placa de silício. Imagens obtidas através de AFM em água (a) após 15 minutos, (b) e (c) após 1 h, (d) após 6 h e (e) após 16 horas de adsorção de CPK sobre silício. Variação da espessura, d, do filme de CPK adsorvido sobre silício a pH 6,8 obtida por AFM medindo-se a altura do filme na fronteira filme-substrato obtida após remoção do parafilme ® (f). (página 66)