Glucose oxidase enzimi substrat, inhibitör ve aktivatörlerine ait kinetik parametrelerin tayini

Abstract

ÖZET Bu çalışmada Aspergillus niger'den elde edilen Glukoz Oksidaz enziminin B-D-glukozu enzimatik olarak yükseltgeme reaksiyonu, aerobik ortamda farklı üç indikatör reaksiyondan faydalanarak spektrofotometrik teknik ile incelendi. Çalışmada inhibitörsüz veya aktivatörsüz ve değişik inhibitor ve aktivatler ile denemeler yapıldı; bunlara ait kinetik parametreler hesaplandı. 25°C da, pH'ı 7,0 olan 0,12 M fosfat tamponunda GOD-FOD- boya (o-dianisidin) metoduyla yapılan denemelerde inhibitörsüz veya aktivatörsüz durumda Kg= 2,°0419xl0 M bulundu. Bu metödla Na2S, NaN02, NaNO3 için rekabet edici (kompetitif) inhibisyon tesbit edildi ve inhibisyonun inhibitörlerin konsantrasyonları ile arttığı görüldü; inhibisyon sabiti Kj_, Na2S için 1,2955x10 M ve NaN02 için 6,3250xl0-4 M ve NaNO3 1, 2030x10` 5 M olarak S-2 - bulundu. ve N-02 asidik ortamda dayanıksız oldukları için, inhibisyon etkileri sadece bu metodla incelenebildi. GOD-POD-boya metoduyla Na3,, Na2SO4 ile yapılan denemelerde esas olmayan aktivasyona uyan sonullar elde edildi ve aktivasyon sabiti KA ; NaN3 için 8,0729xlO-5 M ve Na2S04 için 1, 2069x10-4 M bulundu. 30°C da, pH'ı 6,1 olan 0,84 M fosfat tamponunda I3 fün ölçülmesine dayanan denemelerde inhibitörsüz veya aktivatörsüz durumda Ks - 1,9917x10-4M bulundu. NaNO3'ın inhibitor olarak kullanıldığı durumda kompetitif inhibisyon gözlendi ve inhibis yon sabiti Ki= 1,0674x10-3 M olarak hesaplandı. NaN3 ve Na2SO4 in kullanıldığı denemelerde esas olmayan aktivasyon elde edildi ve aktivasyon sabiti KA ; NaN3 için 8, 0840x10`-5 M ve Na2S04 ıııiçin l,2341x10-4 M bulundu. pH'ı 5,0 olan 0,5 M asetat tamponunda, 30°C da I-, molibdat ve o-tolidin bulunan ortamda yapılan denemelerde inhibitör- KS_ 1,9914*10-4 süz veya aktivat örsüz durumda KS- 1,9914x10 M bulundu. Bu seri denemelerde NaNO, için kompetitif inhibisyon tesbit edildi ve inhibisyon sabiti Ki=1,1733x10 -3 M olarak hesaplandı ; NaN3, NaoSO2, için esas olmayan aktivasyon belirlendi ve aktivasyon sabiti KA, NaN3, için 8,0612x10-5 M ve Na2S04 için l,2371xl0``4 M bulundu. Çalışmada elde edilen sonuçlara göre I-3 ölçümüne dayanan metodun GOD-POD-boya metodundan daha hassas olduğu tesbit edildi, iv

SUMMARY In this study, the enzymic oxidation of /3-D-glucose by Glucose Oxidase (GOD) enzyme obtained from Aspergillus, niger, under aerobic conditions were studied with a spectrophotometry method by the use of three indicator reactions. In the study, various experiments have been carried out without inhibitors and activators, and with different inhibitors and activators, and their kinetic parameters have been calculated. In the experiments carried out by GOD-POD-dye (o-diani- sidine) method at 25°C in 0-,12 M phosphate buffer, pH 7»0, Michaelis-Menten constant Kg was calculated as 2,0419x10 M without inhibitors and activators. It was determined that NapS, NaN02 and NaNO, had competitively inhibited the reaction and it was observed that the inhibition effects have increased with the rising the concentrations of them. The inhibition constant Ki was calculated as 1,2955x10`^ M for NapS, 6,3250x10` M for NaN02 and 1,2030x10 M for NaNO,. The inhibition effect -2 - of S and NOp could only be experimented in this method because these substances are unstable in. an aqueous acidic solution. The results fitted to nonessential activation type were obtained from the experiments carried out with the GOD-POD-dye method for NaN,, NapSO^,. The activation constant KA was calcu lated as 8,0729xl0~5 M for NaN, and l,2069xlO`4 M for Na2S04. At 30°C, 0,84 M fhosphate buffer, pH 6,1, the experiments based on the measurement of I* with the absent of inhibitors or activators, Kg was found as 1,9917x10 M. When NaNO^ was used as inhibitor, competitive inhibition was observed andAIa» (t1 « inhibition constant Ki was calculated as 1,0674x10 ? Mi; In,` the experiments in which NaN, and Na^SO^ were used none s s en--: tial activation was observed and activation constant K^. was found as for KaW^ 8, 0840x10`` 5 M and- Na^SO^ 1, 234-lxlO``4 M. In 0,5 M acetate buffer with pH 5.0, the experiments made under the condition at 30 °C, in which there was I``, mo- lybdate and o-tolxdine Kg was found as 1,9914x10 M. Compet itive inhibition for NaNO, was found in these series experi ments and the inhibition constant Ki was calculated as -4 1*1733x10 M ; nonessential activation for NaN,, NagSO^ was observed and the activation constant K^ were found as 8,0612xl0``5 M for NaNj and l,2371xl0~4 M for NapSO^. According to the results obtained from this study, the method based on the measurement of ZZ was proved that it was more sensitive than the GOD-POD-dye method. M VI