Optimalisasi Waktu Inkubasi dan Konsentrasi Pepsin pada Aktivitas Produksi Serum Anti Tetanus

Tetanus merupakan penyakit yang disebabkan oleh kuman Clostridium tetani. Penyakit ini termasuk mematikan di negara berkembang. Melihat masih tingginya kasus tetanus di negara berkembang serta untuk memberikan pelayanan terbaik dan pengobatan efektif terhadap pasien yang terkena penyakit tetanus, maka diperlukan optimalisasi pada proses pembuatan serum anti tetanus, sehingga dapat meningkatkan kapasitas produksi dan kualitas serum yang dihasilkan. Berdasarkan latar belakang tersebut, penelitian ini bertujuan untuk mengetahui optimalisasi waktu inkubasi dan konsentrasi pepsin pada aktivitas produksi serum anti tetanus. Penelitian ini merupakan penelitian deskriptif kuantitatif dengan teknik pengumpulan data melalui observasi dan studi pustaka. Berdasarkan hasil analisis menggunakan software khusus densitometri Design of Experiment (DoE) dengan metode Sodium Dodecyl Sulphate Polyacrylamide Gel Elektroforesis (SDS PAGE) bahwa konsentrasi optimum didapatkan pada sampel anti tetanus ke-7 dengan konsentrasi pepsin sebesar 0,18 % dalam waktu inkubasi 5 jam.

The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins.Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis.Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.