SANTI NURBAITI
Kelompok Keahlian Biokimia, Fakultas Matematika dan Ilmu Alam ITB.

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Molecular Dynamics Analysis of Thermostable DNA Pol I ITB-1

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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One of the thermostable enzymes, which has been widely used in the biotechnological research, is DNA polymerase. The coding sequence of local DNA Pol I gene from a local thermophilic bacterium, namely DNA Pol I ITB-1, has been cloned, sequenced, and overexpressed. However, study on thermostability of this enzyme is very limited. In the present study, thermostability of the protein was evaluated by thermal unfolding simulation at 300, 400, and 500 K. Our simulation revealed that the secondary and tertiary structures of the protein was not significantly affected by thermal perturbation at 300 K, but they were affected and even gradually unfolded by that perturbation at 400 and 500 K. Evaluation of the root mean square fluctuation (RMSF) of individual residues from the simulation at 400 and 500 K revealed the distribution of the thermostability regions in the protein structure. From the RMSF analysis at 400 K, we found that thermostability of the 3’-5’ exonuclease domain was lower compared to that of the other domains. Where as from the RMSF analysis at 500 K, we found that in each domain of DNA pol I ITB-1 there was a single extraordinary thermostable a-helix which was likely to be the core of each corresponding domain. Thus our simulation provides a thermostability map of DNA Pol I ITB-1. Such information will be very valuable for the next genetic engineering work in determining a mutation target to modify thermostability of this enzyme.

MODIFIKASI SINTESIS NUKLEOTIDA BERTANDA [γ-32 P] ATP

Jurnal Radioisotop dan Radiofarmaka Vol 16, No 1 (2013): JURNAL PRR 2013
Publisher : Jurnal Radioisotop dan Radiofarmaka

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ABSTRAKMODIFIKASI SINTESIS NUKLEOTIDA BERTANDA [Y-32P]ATP.Dalam perkembangan biologimolekul, radionuklida dalam bentuk senyawa bertanda telah digunakan sebagai perunut deoxyribonucleicacid (DNA)/ribonucleic acid (RNA) untuk mendalami berbagai macam proses fisiologi dan patologi. Salahsatu senyawa tersebut adalah nukleotida bertanda fosfor-32 (32P) [γ-32P]-adenosine triphosphate {[γ-32P]-ATP} yang banyak digunakan dalam penelitian biologi molekul. Untuk dapat menunjang penelitian biologimolekul di Indonesia, pada penelitian ini telah dilakukan pembuatan senyawa nukleotida bertanda [γ-32P]-ATP melalui reaksi enzimatis dengan melakukan modifikasi pada metoda sintesisnya menggunakanprekursor DL-glyceraldehyde 3-phosphate, nukleotida adenosine di-phosphate (ADP) dan H332PO4, sertaenzim gliseraldehid 3-phosphat dehidrogense, 3-phosphogliserat-kinase dan laktat dehidrogenase. Pemurnian[γ-32P]-ATP hasil sintesis dengan menggunakan kolom kromatografi DEAE-Sephadex. Dari proses sintesisdan pemurnian yang telah dilakukan berhasil diperoleh [γ-32P]-ATP dengan aktifitas 1,175 mCi dankemurnian radiokimia 99,49%. Dengan berhasilnya dilakukan sintesis dan pemurnian [γ-32P]-ATP, makaPusat Radiosiotop dan Radiofarmaka akan dapat menyediakan nukleotida bertanda dimaksud di atas untukmenunjang penelitian biologi molekul di Indonesia.Kata Kunci: nukleotida bertanda [γ-32P]ATP, sintesis, reaksi enzimatis, pemurnianABSTRACTMODIFICATION OF SYNTHESIS NUCLEOTIDES [Y-32P] ATP.In molecular biology, radionuclidesin the form of radiolabeled compounds have been widely used as deoxyribonucleic acid (DNA) / ribonucleicacid (RNA) tracer in order to explore a wide range of physiological and pathological processes. One of suchcompounds is [γ-32P]-adenosine triphosphate {[γ-32P]-ATP} [γ-32P]-ATP which has been widely used in thebiotechnology research. In order to support the biotechnology research in Indonesia in this project, [γ-32P]-ATP had been synthesized by enzymatic reactions with modifying the method of synthesis using theprecursor DL-glyceraldehydde 3-phosphate, nucleotides Adenosine Diphosphate (ADP) and H332PO4 andenzymes glyceraldehid 3-phosphate dehydrogenase, 3-phosphoglyceryc phosphokinase and lactatedehydrogenase. The purification of the synthesized [γ-32P]-ATP, by using DEAE Sephadex columnchromatography.  The synthesis and purification process that had been performed were able in producing of[γ-32P]-ATP with radioactivity of 1,175 mCi and  radiochemical purity of 99,49%.. Having successfullyprepared the [γ-32P]-ATP and application, in the near future the Radioiotopes and RadiopharmaceuticalsCentre is expected to be able in providing the above-mentioned radiolabeled nucleotide for biotechnologyresearch in Indonesia.Key words : labeled nucleotide [γ-32P]-ATP, synthesis, enzimatic reaction, purification

SINTESA ATP BERTANDA P-32 SEBAGAI PERUNUT BIOLOGI MOLEKUL

Jurnal Radioisotop dan Radiofarmaka Vol 14, No 1 (2011): Jurnal PRR 2011
Publisher : Jurnal Radioisotop dan Radiofarmaka

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SINTESA ATP BERTANDA P-32 SEBAGAI PERUNUT BIOLOGI MOLEKUL. Kebutuhan perunut DNAIRNA dalam perkembangan biolologi molekul di Indonesia saat ini sangat dirasakan. Salah satunya adalah nukleotida bertanda [?32p]_ATP yang banyak digunakan dalam penelitian bioteknologi. Penguasaan teknik sintesa nukleotida bertanda ini akan sangat mendukung penelitian dalam bidang biologi molekul. Sintesa ini dilakukan dengan reaksi enzimatis yang merupakan bagian dan proses glikolisis, dimulai dari fruktosa 1,6-diphosphat, nukleotida ADP dan radioisotop P-32 dengan menggunakan enzim aldolase, gliseraldehid 3-fosfat, 3-fosfogliseratkinase dan laktat dehidrogenase. Pemumiannya dengan menggunakan kolom kromatografi DEAE Sephadex. ATP bertanda yang dihasilkan berada pada Rf 0, dan P32 pada Rf 0,6. Diharapkan bahwa kebutuhan nukleotida bertanda di Indonesia dapat terpenuhi dengan dihasilkannya nukleotida bertanda [?32p]_ATP ini, yang juga merupakan dasar untuk sintesa nukleotida bertanda lainnya. Kata Kunci: Perunut DNA/RNA, Proses glikolisis, Enzimatis, Radioisotop P-32, Nukleotida bertanda 32p]_ATP ABSTRACT SYNTHESIS OF P-32 LABELED ATP FOR MOLECULAR BIOLOGY TRACER. DNA /RNA tracer demmand in the development of molecular biology in Indonesia is currently strongly felt. One of them is labeled nucleotide [?32p]_ATP which is widely used in biotechnology research. Capability of nucleotide synthesis techniques will support research in the field of molecular biology. This synthesis was done by enzymatic reactions which is a part of the glycolysis process, starting from fructose 1,6-diphosphat, ADP nucleotide and P-32 radioisotope by using aldolase enzyme, glyceraldehyde 3-phosphate, 3phosphoglycerate kinase and lactate dehydrogenase. Purification was perf0D11ed by using DEAE Sephadex column chromatography. The labeled ATP and P-32 were observed at Rf 0 and Rf 0.6. It is expected that the demand of labeled nucleotide in Indonesia can be fulfilled by the synthesized labeled nucleotide [?32p]_ATP that is the base for synthesis of other labeled nucleotide. Keywords: DNA/RNA tracer, glycolysis process, enzymatic, P-32 radioisotope, nucleotide labeled [?32p] ATP  

SINTESIS DAN KARAKTERISASI NUKLEOTIDA BERTANDA [a-32P]ATP

Jurnal Riset Kimia Vol 12, No 2 (2019): March
Publisher : Universitas Andalas

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Abstract

The utilization of nuclear technology in health sector with molecular techniques is increasingly developed today, especially in Indonesia.  One of which is nucleotide compound marked with [α-32P]ATP, this compound has been used as tracer for deoxyribonucleic acid (DNA)/ ribonucleic acid (RNA) in the study of various physiological and pathological processes. [α-32P]ATP is synthesized through several stages of continuous reaction in one reaction vessel. It begins with synthesis of [γ-32P]ATP through an enzymatic reaction, using H332PO4 and ADP, and enzymes  of lactate dehydrogenase, 3-phosphoglycerate phosphokinase and glyceraldehide 3-phospho  dehydrogenase; followed by phosphorilation of 3’AMP with T4 polinucleotide kinase enzyme to produce 3’-[5’-32P]ATP. The result is hydrolyzed with nuclease P1 enzyme to produce [5’-32P]AMP. The unreacted [γ-32P] is degraded by the addition of hexokinase enzyme and glucose. At the final stage of the reaction, the [5’-32P]AMP is  phosphorilated using phosphoenol-piruvat, piruvat kinase, and myokinase to produce [α-32P]ATP. The test results show that the every stage of reaction is characterized using TLC method, PEI cellulose paper as stationary phase and KH3PO4 0,5 M pH 3,5 as mobile phase. At the end of reaction, the yield of [α-32P]ATP reaches 71,7%, at Rf = 0,2.