Stability of P-Nitrophenyl Propionate Substrate for Spectrophotometric Measurement of Lipase Activity (Turkish with English Abstract)

Abstract

p-Nitrophenol alconate esters are preferred substrates for spectrophotometric measurement of lipase activity. The method relies on lipolytic release of chromogenic p-nitrophenol (pNP) from the substrates. The study aimed to investigate susceptibility of p-nitrophenyl propionate (pNPP) substrate against non-enzymatic hydrolysis and absorptivity of pNP under various conditions. In general, greater amounts of pNPP were self-hydrolyzed upon increases in storage temperature or duration. Compared to refrigeration temperature, the storage at room temperature for 10 days resulted in ten-fold increase in spontaneous hydrolysis from 0.512 mM pNPP solution. Upon sequential incubation at 30, 60 and 95o C for the corresponding durations of 90, 20 and 5 minutes, the rates of non enzymatic hydrolysis were 0.0014, 0.0154 and 0.0456 absorbans per min, respectively. pNP product absorbans at 404 nm was strongly affected by pH between 6 and 8. Also, increasing buffer capacity from 0.1 to 1.0 M was resulted in at least twofold linear increase in absorbans at 404 or 348 nm. These susceptibilities may be corrected by avoidance of conditions leading to non-enzymatic hydrolysis as well as standardizations of pH and buffer capacity in the assay medium. Finally to minimize minor errors, the enzyme assay should be carried out along with a “parallel control” which contains all components of assay mixture except for the enzyme.

Keywords

• p-nitrophenyl propionate, lipase assay, spectrophotometric method, nonenzymatic hydrolysis

Lipaz Aktivitesinin Spektrofotometrik Yöntemle Belirlenmesinde Çevresel Koşulların P-Nitrofenil Propiyonat Substratının Kararlığına Etkisi

Abstract

Lipaz aktivitesinin spektrofotometrik yöntemle ölçülmesinde p-nitrofenol esterleri tercih edilmektedir. Yöntem, bu substratlardan lipaz hidrolizi ile açık sarı renkli p-nitrofenol (pNP) oluşturulmasına dayanmaktadır. Bu çalışmada, lipaz aktivite ölçümünde kullanılan p-nitrofenil propiyonat (pNPP) substratının kararlılığına ve pNP ürününün ışık soğurmasına çevresel koşulların etkisi incelenmiştir. Bekletme sıcaklığı veya süresi arttıkça, daha fazla oranda pNPP’nin kendiliğinden parçalandığı görülmüştür. On günlük bir depolama sonunda, 4o C’ye kıyasla 20o C’de 10 katı oranında pNPP (0.512 mM) kendiliğinden parçalanmıştır. Birbirini takip edecek şekilde 30, 60 ve 95o C’lerde sırasıyla 90, 20 ve 5 dakika inkübasyon süreleri, dakikada 0.0014, 0.0154 ve 0.0456 absorbans artışlarına neden olmuştur. Öte yandan, oluşan pNP ürününün 400nm civarında göstereceği absorbans değeri pH 6-8 aralığında ortam pH’sına oldukça duyarlı iken; tampon kapasitesi arttıkça (0.1-1.0 M) pNP ürünün gösterdiği absorbans (404 veya 348 nm) doğrusal artarak en az 2 kat düzeyine çıkmıştır. Bu duyarlılıklar, substratın kendiliğinden hidrolizine neden olacak koşulların azaltılması, pH ve tampon kapasitesinin standardizasyonu ile minimize edilebilmektedir. Buna ilave olarak, enzim hariç tepkime karışımının tüm bileşenlerini içeren bir “paralel kontrol” eşliğinde denemelerin yürütülmesiyle küçük hata kaynaklarının çoğunlukla sınırlandırılabileceği tespit edilmiştir.

Keywords

• p-nitrofenil propiyonat, lipaz aktivitesi, spektrofotometrik yöntem, kendiliğinden parçalanma

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