A procedure for the purification of uricase from porcine liver is described, utilising the technique of bioaffinity chromatography as a major purification step. Bioaffinity support is prepared by coupling of urate to bisoxirane-activated Sepharose 4B. Purified uricase shows a single protein band corresponding to the activity band, when applied to polyacrylamide disc-gel electrophoresis. A single protein band but no activity band is obtained by SDS-acrylamide disc-gel electrophoresis. The enzyme has a pH optimum in the range of 8.9-9.1, with a Vmax of 12.6 U.mg^-1, a Km of 1 X 10^-5M and a molecular weight of 13 x 10⁴. Each enzyme molecule comprises 4 subunits of molecular weight 3 32-34 X 10³ each. Nylon tube is directly activated by alkaline glutaraldehyde solution to generate reactive centres for enzyme immobilisation. The optimal conditions for activation are studied. Purified uricase is immobilised to PEI-glutaraldehyde-nylon tube with about 20% activity retention. The derivatised enzyme has a pH optimum in the range of 9.0-9.2 and a Km of about 4 times that of the soluble enzyme. Immobilised uricase is incorporated into a continuous flow Auto-Analyser for use in the automated analysis of serum urate. For this purpose, the immobilised enzyme shows good storage and operational stability. Linear calibration plots can be obtained for a urate range of 2-20 mg.100ml^-1the method exhibiting a high degree of precision and accuracy. The results obtained also compare favourably with an established method of urate assay which employs soluble uricase.

Disponibiliza??o do trabalho em conte?do parcial, conforme Termo de Autoriza??o. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-03-27T19:34:05Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) fernanda_souza_paula_parcial.pdf: 515579 bytes, checksum: f72e2289acf5068c063e2bd00f0fefc3 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-04-24T16:35:34Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) fernanda_souza_paula_parcial.pdf: 515579 bytes, checksum: f72e2289acf5068c063e2bd00f0fefc3 (MD5) / Made available in DSpace on 2017-04-24T16:35:34Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) fernanda_souza_paula_parcial.pdf: 515579 bytes, checksum: f72e2289acf5068c063e2bd00f0fefc3 (MD5) Previous issue date: 2017 / O trabalho investiga a utiliza??o de plataformas eletroqu?micas contendo filmes polim?ricos derivados do ?cido 4-aminosalicico (4-AMS) para imobiliza??o da enzima Urato oxidase (UOx) visando aplica??o na quantifica??o de ?cido ?rico (AU) em amostras de urina. Investigou-se a eletrodeposi??o do 4-AMS pelas t?cnicas de voltametria c?clica (VC) e cronoamperometria (CA) sobre eletrodos de grafite (EG). Por VC foram realizados 100 ciclos de potencial na faixa de -0,25 a 1,25 V ? 50 mV/s em solu??o 2,50 mM do mon?mero preparado em H2SO4 0,50 M. Utilizando a CA, a eletropolimeriza??o foi realizada a potencial constante de +0,928 V durante 5600s no mesmo meio reacional utilizado na VC. O poli(4-AMS) obtido por VC e CA mostrou dois pares redox, os quais est?o relacionados a eletroatividade do filme polim?rico, na regi?o de potencial de +0,50/+0,40 V. Contudo, maiores valores de Ipa e Ipc foram obtidos para os eletrodos modificados por VC, sugerindo que estes filmes s?o mais eletroativos. A deposi??o do azul da Pr?ssia (AP), mediador da rea??o de per?xido, foi investigada sobre os EG, com posterior modifica??o com poli(4-AMS). Notou-se que a presen?a do AP n?o altera o perfil voltam?trico da eletropolimeriza??o do 4-AMS. Contudo, quando comparada com a eletropolimeriza??o somente nos EG, obteve-se filmes mais resistivos e com menor eletroatividade. Analisando as propriedades eletroqu?micas e morfol?gicas, por VC conseguiuse filmes mais uniformes, com maior quantidade de material depositado e maior eletroatividade. A eletropolimeriza??o foi realizada tamb?m sobre eletrodos impressos de grafite contendo azul da Pr?ssia (EI/AP), onde posteriormente imobilizou-se 5 U da UOx, e o biossensor foi acoplado a uma c?lula de fluxo num sistema de an?lise por inje??o em fluxo (FIA) de linha ?nica. A vaz?o e o volume da al?a de amostragem foram otimizados em 2,10 mL/min e 200 ?L, repectivamente. O valor de pH da solu??o do analito foi otimizado em 8,27. Medidas de reprodutibilidade mostraram desvio padr?o de 2,15% (n=10). O biossensor respondeu linearmente para AU na faixa de 1,0 x 10-5 a 2,0 x 10-4 M, com limite de detec??o de 3,0 ?M. Amostras de urina foram dilu?das (1:10) e injetadas diretamente no biossensor. A reposta foi reprodut?vel mostrando baixo desvio padr?o para as medidas, e valores encontrados dentro da faixa esperada para o analito em amostras de urina. Testes de adi??o e recupera??o mostraram valores de 97,35% (?2,43). O biossensor mostrou-se bastante promissor para a proposta do trabalho, apresentando resultados muito satisfat?rios para as an?lises e par?metros investigados. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / This work investigates the use of electrochemical platforms containing polymeric films derived from 4-aminosalicylic acid (4-ASA) modified with the enzyme Urate oxidase (UOx) for quantification of uric acid (UA) in urine samples. The electrodeposition of 4-ASA was investigated through Cyclic Voltammetry (CV) and Chronoamperometry (CA) on graphite electrodes (GE). 100 cycles were performed in the range of -0.25 to 1.25 V at 50 mV/s in 2.50 mM monomer solution prepared in 0.50 M H2SO4. Using CA, the deposition was performed at a potential of +0.928 V for 5600 s in the same CV reaction medium. The poly(4-ASA) showed two redox pairs related to the electroactivity of the polymeric film in the potential range of + 0.50 /+ 0.40 V. However, higher values of Ipa and Ipc were obtained for the electrodes modified through CV, suggesting that these films are more electroactive. The deposition of Prussian blue (PB), mediator of the peroxide reaction, was investigated on the GE with subsequent modification with poly (4-ASA). It was observed that the presence of PB does not alter the voltammetric profile of 4-ASA electropolymerization. However, when compared with the electropolymerization in bare GE, more resistive films were obtained with lower electroactivity. Analyzing the electrochemical and morphological properties through CV, more uniform films were obtained, with more material deposited and greater electroactivity. The electropolymerization of poly(4-ASA) was also conducted on screen printed electrodes containing Prussian Blue (SPE/PB), with subsequent immobilization of 5U of Uox. This biosensor was coupled to a flow cell in a Flow Injection Analysis (FIA) system of single line. The flow rate and the sampling loop volume were optimized at 2.10 mL/min and 200 ?L, respectively. The pH value of the analyte solution was optimized at 8.27. Reproducibility measures showed a standard deviation of 2.15% (n = 10). The biosensor responded linearly to UA in the range of 1.0 x 10-5 to 2.0 x 10-4 M, with a detection limit of 3.0 ?M. Urine samples were diluted (1:10) and directly injected over the biosensor. The response was reproducible with low standard deviation and values found within the range expected for the analyte in urine samples. Addition and recovery tests showed values of 97.35% (?2.43). The biosensor is very promising for the work proposal, presenting very satisfactory results for the analyzes and investigated parameters.