Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 `M) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 `M), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 `M) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 + or - 0.8 `M and k cat = 48.4 + or - 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 + or - 10, 1,098 + or - 91, 38.6 + or - 5.2 and 37,340 + or - 5,400 `M, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 + or - 92.8 and 310,500 + or - 38,600 `M, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds

Kinetic characterization of rat tissue kallikrein using NÓ-substituted arginine 4-nitroanilides and NÓ-benzoyl-L-arginine ethyl ester as substrates

Hydrolysis of seven N(alpha-substituted L-arginine 4-nitroanilides: henzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N(alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 muM for p-nitroanilides and 50 to 190 muM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 muM for p-nitroanilides and 200 to 1800 muM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 ñ 2 muM; Vmax 10.42 ñ 0.28 muM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 ñ 2 muM; Vmax = 3.21 ñ 0.11 muM/min), while Tos-Arg-Nan (Km = 29 ñ 2 muM; Vmax, = 0. 10 ñ 0.002 muM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 ñ 15 muM; Vmax = 121.3 ñ 7.6 muM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Soro-controle de origem bovina e seu emprego no controle de qualidade em bioquímica clínica / Control serum of bovine origin and its usage in quality control in clinical biochemistry

Os autores apresentam estudo de um soro-controle preparadoa partir de material de origem bovina e seu desempenho no controle de qualidade em bioquimica clinica. Esse soro foi preparado na proporção de 6 partes de soro e 4 partes de glicerol adicionando-se 0,1g de azida sódica para cada 100ml. A análise estatística dos resultados obtidos ao longo de oito meses mostrou uma boa estabilidade dos constituintes bioquimicos quando comparados com os valores previamente estabelecidos. As vantagens do uso de soro-controle de origem bovina no laboratório de análises clínicas compreendem a facilidade de obtenção de grandes volumes e proporciona ao analista, segurança em sua manipulação, evitando maiores riscos de contaminação por agentes infecciosos especificamente quanto à hepatite B e AIDS.

Nova formulaçao para o preparo do tampao alcalino utilizado na dosagem de creatinina no sangue e urina metodo de Heinegard-Tiderstrom modificado / New formulation for the preparation of the alkaline buffer used in the determination of blood's and urine's creatinine - modified Heinegard-Tiderstrom's method

Neste trabalho e apresentada uma nova formulaçao para opreparo do tampao alcalino empregado no Metodo de Heinegard-Tiderstrom modificado por LOPES et alii, 1984, para a dosagem de creatinina no sangue e urina. A modificaçao foi proposta com a finalidade de se obter uma metodologia que facilitasse o preparo do tampao alcalino, garantindo a nao precipitaçao em baixas temperaturas, alem da reduçao do custo. A nova formulaçao do tampao alcalino foi avaliada em comparaçao com o reagente de Heinegard-Tiderstrom mod.(8), atraves da dosagem de 100 (cem) amostras de soro com variadas concentraçoes de creatinina. A analise dos resultados demonstrou uma excelente correlaçao linear, sugerindo que este reagente preenche todos os requisitos necessarios ao seu emprego na dosagem de creatinina, apresentando ainda as vantagens acima citadas