Purification and some properties of Candida albicans DNA polymerases.

DNA polymerases of Candida albicans were purified to near homogeneity. Three well distinguished peaks of DNA polymerase activity (Enzyme I, II and III respectively) were obtained by DEAE-Sephacel chromatography. This purification step was followed by column chromatographies on Sepharose 6B and denatured DNA-cellulose. The enzymes' molecular mass and biochemical properties, including their inhibition by aphidicolin, were studied. Molecular mass was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and was found to be 110 kDa for Enzyme I, 80 kDa for Enzyme II and 50 kDa for Enzyme III.     

Purification and partial characterisation of tentatively classified acid phosphatase from the earthworm Eisenia veneta

In order to use leakage of lysosomal acid phosphatase (AP) as a biomarker of stress to earthworms, more information about AP’s in earthworms are needed. This paper describes the details about tentatively classified APs in the earthworm Eisenia veneta. Two isoenzymes (enzyme I and II) of acid phosphatase (AP) and one alkaline phosphatase (enzyme III) from the earthworm E. veneta were separated by gel filtration. All three enzymes were further purified and concentrated on a Con A Sepharose 4B column. Enzyme I was inhibited by tartrate, showed an optimal pH range between 4.0 and 5.0 and was assumed to be of lysosomal origin. Enzyme II was the major enzyme showing the highest activity of the three enzymes. It was expected to be a lysosomal AP under physiological conditions. Enzyme II had a molecular mass 113 kDa and was composed of apparently identical polypeptide chains of 36 kDa each. This enzyme was inhibited by tartrate, showed an optimal pH in the range 6.0–7.5 and was slowly degraded at temperatures above 40°C. Enzyme III is not inhibited by tartrate and has a pH-optimum >9. The subcellular location under physiological conditions was assumed to be the cytosol.     

Aryl alcohol oxidases from the white-rot basidiomycete Pleurotus ostreatus

 Three aryl alcohol oxidases (AAOs; EC 1.1.3.7) I, II, and III from the culture filtrate of a strain of white-rot fungus Pleurotus ostreatus were purified by multistep chromatography. Each of the purified AAOs I, II, and III had the same molecular masses of 70 kDa and 72 kDa on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Their optimum temperature was 40°C, but their optimum pHs differed slightly. The N-terminal amino acid sequence of AAOs I, II, and III was determined to be Ala-Asp-Lys-Asp-Tyr-Ile-Val-Val-Gly-Ala, which showed significant similarity to those of Pleurotus eryngii (80% identity) and Pleurotus ostreatus Florida (60% identity). Received: May 30, 2002 / Accepted: July 10, 2002 Acknowledgment This work was supported in part by a Grant-in-Aid for the Encouragement of Young Scientists (no. 12760117) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:K. Okamoto     

Human 170 kDa and 180 kDa Topoisomerases II Bind Preferentially to Curved and Left-Handed Linear DNA

Abstract

The binding activities of the 170 kDa and the 180 kDa human topoisomerases II (topo IIa and topo IIβ) to linear DNA fragments with different degrees of curvature were characterized. In gel retardation experiments it was shown that both forms of the enzyme bind preferentially to a curved 287 bp fragment, forming a detectable stable complex. The affinity for straight DNA fragments of similar length is significantly lower. Both a commercially available topo IIa, isolated from placenta, and topo IIα and topo IIβ purified from nuclear extracts of the Namahva lymphoma tissue culture line gave similar results. The effects of double-stranded poly[d(A-T)], poly[d(G-C)], supercoiled plasmid DNA and linear Z-DNA on the topo II- complex with curved DNA were analyzed in competition experiments. The hierarchy of affinities of the 180 kDa topo IIβ for these DNAs has the order: linear left-handed DNA > supercoiled DNA ? curved DNA ? poly[d(A-T)] ? poly[d(G-C)]. The 170 kDa topo IIa binds with similar affinity to curved DNA and linear Z-DNA ? supercoiled DNA ? linear B- DNA The data imply that human topoisomerase II binding is more sensitive to DNA secondary structure than to DNA sequence per se. The ability of the enzyme to preferentially recognize a wide variety of sequences in unusual secondary structures suggests a mode of targeting the enzyme in vivo to regions of high negative supercoiling.     

DNA polymerases from bakers' yeast.

Two DNA polymerases are present in extracts of commercial bakers' yeast and wild type Saccharomyces cerevisiae grown aerobically to late log phase. Yeast DNA polymerase I and yeast DNA polymerase II can be separated by DEAE-cellulose, hydroxylapatite, and denatured DNA-cellulose chromatography from the postmitochondrial supernatants of yeast lysates. The yeast polymerases are both of high molecular weight (greater than 100,000) but are clearly separate species by the lack of immunological cross-reactivity. Analysis of associated enzyme activities and other reaction properties of yeast DNA polymerases provides additional evidence for distinguishing the two species. Enzyme I has no associated nuclease activity but does carry out pyrophosphate exchange and pyrophosphorolysis reactions, and has an associated 3'-exonuclease activity. Enzyme I does not degrade deoxynucleoside triphosphates and cannot utilize a mismatched template. Enzyme II does carry out a template-dependent deoxynucleoside triphosphate degradation reaction and can excise mismatched 3'-nucleotides from suitable template systems. Earlier studies have shown that both Enzyme I and Enzyme II are inhibited by N-ethylmaleimide. The yeast enzymes are not identical to any known eukaryotic or prokaryotic DNA polymerases. In general, Enzyme I appears to be most similar to eukaryotic DNA polymerase alpha and Ezyme II exhibits properties of prokaryotic DNA polymerases II and III.     

Post-translationally modified human lens crystallin fragments show aggregation in vitro

BackgroundCrystallin fragments are known to aggregate and cross-link that lead to cataract development. This study has been focused on determination of post-translational modifications (PTMs) of human lens crystallin fragments, and their aggregation properties.MethodsFour crystallin fragments-containing fractions (Fraction I [～3.5 kDa species], Fraction II [～3.5–7 kDa species], Fraction III [～7–10 kDa species] and Fraction IV [>10–18 kDa species]), and water soluble high molecular weight (WS-HMW) protein fraction were isolated from water soluble (WS) protein fraction of human lenses of 50–70 year old-donors. The crystallin fragments of the Fractions I–IV were separated by two-dimensional (2D)-gel electrophoresis followed by analysis of their gel-spots by mass spectrometry. The Fractions I–IV were examined for their molecular mass, particle-diameters, amyloid fibril formation, and for their aggregation by themselves and with WS-HMW proteins.ResultsCrystallin fragments in Fractions I–IV were derived from α-, β- and γ-crystallins, and their 2D-gel separated spots contained multiple crystallins with PTMs such as oxidation, deamidation, methylation and acetylation. Crystallin fragments from all the four fractions exhibited self-aggregated complexes ranging in M_r from 5.5×10⁵ to 1.0×10⁸ Da, with diameters of 10–28 nm, and amyloid fibril-like formation, and aggregation with WS-HMW proteins.ConclusionThe crystallin fragments exhibited several PTMs, and were capable of forming aggregated species by themselves and with WS-HMW proteins, suggesting their potential role in aggregation process during cataract development.General significanceCrystallin fragments play a major role in human cataract development.     

On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in rat liver.

Evidence is presented for the occurrence of two different non-specific nucleotide-sugar hydrolases in rat liver and other rat tissues. These two enzymes (I and II) were separated by chromatography on a 5'-AMP-aminohexyl-Sepharose column. Enzyme I is most probably identical with phosphodiesterase I (EC 3.1.4.1). Enzyme II appeared to be identical with an enzyme described in literature as 'CMP-sialic acid hydrolase' [Kean & Bighouse (1974) J. Biol. Chem. 249, 7813-7823], since almost all activity with CMP-N-acetylneuraminate as substrate was recovered in this enzyme fraction. CMP-N-acetylneuraminate was a poor substrate for Enzyme I, whereas deoxythymidine-5'-p-nitrophenyl phosphate and all nucleoside-diphosphosugars tested were good substrates for both Enzyme I and II. Therefore it is suggested that CMP-N-acetylneuraminate is used as an additional substrate to discriminate between the activities of Enzyme I and II in homogenates or membrane preparations. The various substrates appeared to be competitive inhibitors of each other, suggesting that, in each enzyme preparation, only one enzyme is responsible for the hydrolysis of the various substrates. The dissimilar properties of the two enzymes are substantiated by studying the subunit molecular masses (Enzyme I, 125 kDa; Enzyme II, 50-55 kDa), the sensitivity towards Triton X-100, Sarkosyl and sodium dodecyl sulphate and towards trypsin treatment. It is discussed whether the alpha-N-acetylglucosamine phosphodiesterase described by Varki & Kornfeld [(1981) J. Biol. Chem. 256, 9937-9943] is identical with one of the nucleotide-sugar hydrolases described here.     

Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the fungus Podospora anserina

Three DNA-dependent RNA polymerases have been isolated and partially purified from the mycelium of the fungus Podospora anserina. Separated by DEAE-Sephadex chromatography, they have been designated RNA polymerases I, II, and III according to their order of elution. Their catalytic properties and alpha-amanitin sensitivity are in agreement with those of the homologous enzymes found in other eukaryotic organisms. The three enzymes exhibit rather sharp monophasic ammonium sulfate dependence with optima which are, respectively, 0.035 M, 0.050 M, and 0.075 M. Enzyme I has the largest Mn2+/Mg2+ activity ratio, shows a marked preference for native DNA, and is insensitive to alpha-amanitin. Enzyme III uses poly(dA-dT) in preference to native DNA as template and is only partially sensitive to alpha-amanitin. Enzyme II is sensitive to alpha-amanitin, but high concentrations of the toxin are required for inhibition compared to other eukaryotic class II enzymes. Three similar RNA polymerases with comparable levels of activity were found in the temperature-dependent VR strain when cellular incompatibility, leading to a rapid cessation of RNA synthesis, was induced.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase

The phytochrome gene (phyCer) of the moss Ceratodon purpureus was isolated and characterized. phyCer is composed of three coding exons: exon I of 2035 bp, exon II of 300 bp and exon III of 1574 bp. The deduced polypeptide encoded by exon I and II exhibits substantial sequence homology to the conserved NH₂-terminal chromophore domain of known phytochromes. In contrast, the COOH-terminal polypeptide encoded by exon III shows no sequence homology to any phytochrome molecule. phyCer most likely represents a single-copy gene and is expressed in a light-independent manner. From the DNA sequence analysis it can be deduced that the PhyCer polypeptide is composed of 1303 amino acids (including the starting Met) which predicts a molecular mass for PhyCer of 145 kDa. The polypeptide encoded in exon III exhibits striking homology within the 300 carboxy-terminal amino acids to the catalytic domain of protein kinases. The carboxy terminus of PhyCer was found to be most homologous to protein-tyrosine kinases of Dictyostelium discoideum and to the products of retroviral oncogenes which belong to the Raf-Mos serine/threonine kinase family. From the hydropathy profile PhyCer appears to be a soluble protein. The predicted structure suggests that PhyCer represents a soluble light-sensor protein kinase which is linked with a cellular phosphorylating cascade.     

Properties of hemicellulases of the enzyme complex from Trichoderma longibrachiatum

Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained earlier based on the strain 1 Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pI 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, β-xylosidase (β-XYL; 80 kDa, pI 4.5) and α-L-arabinofuranosidase I (α-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of preparations Celloviridin G20x and Xybeten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (α-L-AF I or β-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with α-L-AF I.     

Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca)

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (K_i) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human neutrophil elastase inhibitors described in a mollusk.     

胆道支架置入联合介入化疗对恶性胆道梗阻患者肝功能及预后的影响

目的:研究胆道支架置入联合介入化疗对恶性胆道梗阻患者肝功能及预后的影响,为临床治疗提供依据。方法:选取2013年2月到2015年2月我院收治的恶性胆道梗阻患者90例,按照随机数字表法将患者分为Ⅰ组、Ⅱ组和Ⅲ组,每组30例,Ⅰ组给予胆道支架置入联合介入化疗,Ⅱ组给予单纯胆道支架置入,Ⅲ组给予保守治疗,比较三组治疗前、后肝功能、并发症、支架通畅率及生存期。结果:治疗前三组谷草转氨酶(AST)、谷丙转氨酶(ALT)、γ-谷氨酰转移酶(r-GT)比较无统计学意义(P0.05),治疗后Ⅰ组和Ⅱ组AST、ALT和r-GT均显著改善,与治疗前和Ⅲ组比较差异具有统计学意义(P0.05),且I组显著优于Ⅱ组,比较差异具有统计学意义(P0.05),Ⅲ组治疗后AST、ALT和r-GT与治疗前比较差异无统计学意义(P0.05);Ⅰ组、Ⅱ组和Ⅲ组并发症发生率比较无统计学意义(P0.05);Ⅰ组术后3个月、6个月和12个月支架通畅率均显著高于Ⅱ组,比较差异具有统计学意义(P0.05);I组生存期显著高于Ⅱ组和Ⅲ组,Ⅱ组高于Ⅲ组,比较差异具有统计学意义(P0.05)。结论:胆道支架置入联合介入化疗治疗恶性胆道梗阻具有较好效果,能明显改善患者肝功,延长患者生存期。     

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30 kDa; serotype II, 38 kDa; serotype III, 34 kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores—< 51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.     

Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.     

Xylanase Activity of Phanerochaete chrysosporium

Xylan-degrading enzymes were induced when Phanerochaete chrysosporium was grown at 30°C in shake flask media containing xylan, Avicel PH 102, or ground corn stalks. The highest xylanase activity was produced in the corn stalk medium, while the xylan-based fermentation resulted in the lowest induction. Analytical and preparative isoelectric focusing were used to characterize xylanase multienzyme components. Preparative focusing was performed only with the cultures grown on Avicel and corn stalk. Of over 30 protein bands separated by analytical focusing from the Avicel and corn stalk media, three main groups (I, II, and III) of about five isoenzymes each showed xylanase activity when a zymogram technique with a xylan overlay was used. Enzyme assays revealed the presence of 1,4-β-endoxylanase and arabinofuranosidase activities in all three isoenzyme groups separated by preparative isoelectric focusing. β-Xylosidase activity appeared in the first peak and also as an independent peak between peaks II and III. Denatured molecular masses for the three isoenzyme groups were found to be between 18 and 90 kDa, and pI values were in the range of 4.2 to 6.0. β-Xylosidase has an apparent molecular mass of 20, 30, and 90 kDa (peak I) and 18 and 45 kDa (independent peak), indicating a trimer and dimer structure, respectively, with pI values of 4.2 and 5.78, respectively. Three more minor xylanase groups were produced on corn stalk medium: a double peak in the acidic range (pI 6.25 to 6.65 and 6.65 to 7.12) and two minor peaks in the alkaline range (pI 8.09 to 8.29 and 9.28 to 9.48, respectively). The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on corn stalk media was more complex than that of culture supernatants from cells grown on cellulose. The pH optima of the three major xylanase groups are in the range of pH 4 to 5.5.     

Isolation and immunological characterization of three highly purified serine proteinases from Antarctic krill (Euphausia superba)

Summary Three individual serine proteinases (I, II, III) originating from Antarctic krill (E. superba) were separated and highly purified using a combination of affinity and high resolution ion exchange chromatography. Each enzyme showed a single protein band (30 000 Daltons) in sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis indicating high purity and identical molecular weights. Moreover, each enzyme demonstrated one immunoprecipitate on crossed immunoelectrophoresis (two-dimensional agarose gel electrophoresis) using polyclonal rabbit antibodies which confirmed the high purity of the individual enzymes. A mixture of the three enzymes (I, II, III) revealed two immunoprecipitates, not one or three which should have been the case for identical or non-identical immunological properties. Double immunodiffusion test according to Ouchterlony exhibited immunological identity between enzyme II and III. Enzyme I showed only partial identity with II/III. These findings correlated well with biochemical data on the three serine proteinases. Enzyme I is able to liberate free amino acids from polypeptides in comparison with enzyme II and III (classical true endopeptidases), which do not. We suggest that these unique biochemical properties also have their immunological counterpart expressed as other antigenic determinants of the molecular structure.     

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate