PMC:101390 / 11173-24153 JSONTXT

{"project":"2_test","denotations":[{"id":"11914155-6953966-8561970","span":{"begin":196,"end":197},"obj":"6953966"},{"id":"11914155-3465718-8561971","span":{"begin":317,"end":319},"obj":"3465718"},{"id":"11914155-9210347-8561972","span":{"begin":355,"end":356},"obj":"9210347"},{"id":"11914155-6953966-8561973","span":{"begin":627,"end":628},"obj":"6953966"},{"id":"11914155-9210347-8561974","span":{"begin":629,"end":630},"obj":"9210347"},{"id":"11914155-1276392-8561975","span":{"begin":631,"end":633},"obj":"1276392"},{"id":"11914155-1368208-8561976","span":{"begin":3350,"end":3352},"obj":"1368208"},{"id":"11914155-9210347-8561977","span":{"begin":5208,"end":5209},"obj":"9210347"},{"id":"11914155-6953966-8561978","span":{"begin":7358,"end":7359},"obj":"6953966"},{"id":"11914155-1645934-8561979","span":{"begin":11041,"end":11043},"obj":"1645934"},{"id":"11914155-1952054-8561980","span":{"begin":11075,"end":11077},"obj":"1952054"},{"id":"11914155-1368208-8561981","span":{"begin":12610,"end":12612},"obj":"1368208"},{"id":"11914155-8001397-8561982","span":{"begin":12909,"end":12911},"obj":"8001397"}],"text":"Use of Triton X-114 for extraction and purification of COX\nIn most of the available wild producer strains, COX behaves as a cell-linked enzyme, which is particularly true in the genus Rhodococcus[7,8,23]. Significant levels of extracellular COX have been described to be produced by the pathogenic species R. equi[10,11,24,25] and also by R. erythropolis[9] and Rhodococcus sp. [8,24] in certain culture conditions.\nSeveral authors have investigated the ability of different detergents to disrupt lipid-protein associations and to release cell-linked COX in its native state. The use of Triton X-100 has been largely accepted [7,9,12,26,27] but other polyoxyethylene type non-ionic detergents whose cloud point is in the biocompatible range can be used for COX solubilization and purification [17].\nTable 2 shows the amount and the percentage of cell-linked COX extracted by either Triton X-100 or Triton X-114 at several detergent concentrations. COX could be extracted from cells and solubilized by both detergents at 1% w/v. The % of recovery is of the same order as previously reported for these detergents [17]. The increase of the Triton X-114 concentration from 1 to 3% w/v resulted in concomitant increases of 1.5-fold the total activity, 2.6-fold the specific activity and a % of recovery above 90%. A Triton X-114 concentration as high as 6% w/v does not improve significantly the recovery nor the specific activity. Phase separation was further induced to gain COX purity in extracts and in the broth independently.\nTable 2 Cell-linked COX extraction by Triton X-100 and Triton X-114 detergents.\nDETERGENT COX activity (UE/ml) % recovery COX activity (UE/mg Prot)\nTRITON X-1001% 234.2 59.3 1306.9\nTRITON X-114 1% 243.4 61.6 566.9\nTRITON X-114 2% 274.5 69.5 860.5\nTRITON X-114 3% 363.8 92.1 1466.9\nTRITON X-114 6% 365.0 92.4 1674.3 The cell-free extract of Triton X-114 was subjected to phase separation as such. The culture broth was first supplemented with Triton X-114, well dissolved at 4°C and then warmed up to induce detergent phase separation. Figures 2a and 3a show the distribution of enzyme activity in each phase (detergent-depleted and -rich) for each of the COX sources (cells extract and culture broth) respectively.\nFigure 2 Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of culture broth supplemented with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity.\nFigure 3 Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of cell extracts done with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity. As Triton X-114 concentration is increased, COX partitions towards the detergent rich phase, increasing its specific activity (Figures 2b and 3b) thus resulting in enzyme purification and also in enzyme concentration since the volume of the detergent-rich phase is much lower than the initial volume. The 1% concentration of detergent was an exception to this rule since COX partitioned toward the depleted phase under our working conditions. Partitioning of commercial COX in buffers containing 1% Triton X-114 occurred toward the rich phase and was very influenced by the buffer concentration [16]. Therefore, it seems that the composition of phase separation media is extremely important to the partitioning of particular proteins.\nThe purification was made evident by running samples of COX from cells and culture broth in SDS-PAGE gels. Figure 4 shows that in both cases the detergent-rich phase was enriched in some proteins, including COX, whereas the depleted phase showed other different protein bands.\nFigure 4 SDS-PAGE of COX fractions using 3% Triton X-114 for extraction, purification and concentration, (a) Cell extracts: lane 1, Mw markers; lane 2, commercial COX; lane 3, total extracted proteins; lane 4, proteins in detergent depleted phase; lane 5, proteins in detergent rich phase, (b) Culture broth: lane 1, Mw markers; lane 2, commercial COX; lane 3, proteins in detergent rich phase; lane 4, total proteins in culture broth; lane 5, proteins in detergent depleted phase. Arrows indicated the COX band. An exceptional result was obtained when performing COX purification from the culture broth supplemented with a 6% w/v Triton X-114. The total activity recovered after phase separation was ca. 3.5-fold that measured in the broth before phase separation. This result suggests that soluble COX produced by the culture is not fully active and that it can be activated by a treatment with 6% Triton X-114 but not with 4% or less. Further increase of Triton X-114 concentration results in no improvement with respect to 6% (results not shown). This phenomenon was not observed with COX extracted from cells, therefore the enzyme most likely exists in a fully active form in the cells.\nWe have shown previously that cell-linked and soluble COX from the same strain are almost indistinguishable as judged by some enzymatic properties such as kinetic parameters, electrophoretic mobility of the native active enzyme and thermostability [9]. Now we show evidence of a differential characteristic of soluble COX as compared to cell-linked: the activation by 6% Triton X-114.\nThe observed phenomenon accepts in principle several explanations: (i) all the soluble COX molecules become activated by 3.5-fold due to a detergent effect on the protein conformation, (ii) a fraction of soluble COX is active and a fraction 3.5-fold larger is fully inactive, but can be activated due to a detergent effect on the protein conformation, (iii) an inhibitor is removed as a consequence of detergent treatment. From the first hypothesis it could be expected some difference between both enzyme forms at least at kinetic level, which we did not observe in previous studies, although it cannot be discarded. The inhibitor hypothesis is perhaps less likely since the activation effect might have been observed at all concentrations of Triton X-114 and gradually. The second hypothesis may be the most likely according to our previous results since we characterize only active enzyme and not the enzyme protein. In that case it may be hypothesized that there is an active form of COX able to both interact with components of the cell membrane or the cell wall to remain cell-linked, and to stay soluble in the culture broth, and there is an inactive form which is soluble in the culture broth. Reversion of inactive to active is induced by a high detergent concentration, which may provide an environment resembling that of cell membranes or cell walls. The active soluble form secreted by bacterial cells might eventually and reversibly turn into inactive soluble COX. When detergent concentration of the 6% rich phase was lowered by dilution the specific activity did not change, therefore the conversion of active to inactive must be very slow, that is, the existing enzyme forms are not in equilibrium. In any case, the characterization of this activation phenomenon requires further studies that are now under progress in our laboratory.\nThe enzymatic properties exhibited by COX have been shown to depend on the method of extraction from cells, either with Triton X-100, with buffer or with trypsin [7]. The extracted enzyme could be interconverted from one to another with appropriated treatments such as addition or removal of 0.5% Triton X-100. So they showed evidence for the existence of different isoforms of COX from Nocardia rhodochrous (renamed as Rhodococcus rhodochrous). These authors also pointed out that no phospholipids were co-extracted even when using Triton X-100 and suggested that cell-linked enzyme is anchored through a hydrophobic tail that interacts with naturally occurring surfactants of the cell wall.\nTaking that work into account and our own results it may be proposed that COX shed from cell walls aided by bacterial surfactant solubilization becomes the extracellular enzyme and so its level might be related with the production of bacterial surfactants.\nThe effect of Triton X-114 phase separation step on the purification and concentration of COX is summarized in Table 3. Taking together purification, concentration and % of recovery, the best results were obtained when using Triton X-114 at 3% w/v for the cell-linked and at 6% for the extracellular COX. Minuth et al. [17,18] achieved 5-fold purification and 4-fold concentration adding the detergent pentaethyleneglycol mono n-dodecyl ether (C12EO5) to an non-clarified culture of Nocardia rhodochrous. Our results, 11.6-fold purification and 20.3-fold concentration indicate that Triton X-114 should be highly recommended for use with a clarified culture broth of an extracellular COX producer strain.\nTable 3 Purification and concentration of COX during Triton X-114 phase separation.\nCells extract of 3%Triton X-114 w/v Culture broth with 6% Triton X-114 w/v\nPurification fold 1.67 11.6(2.52)b\nConcentration factor 2.56 20.3 (4.38)b\n% recovery 76 (70)a 312 (65)b\naRecovery in this step and, in parenthesis, with respect to cells. b Figures in parenthesis correspond to purification, concentration and recovery in this step as if no activation occurred, calculated on the basis of remaining activity in the upper detergent-depleted phase The quantification of COX partitioning in the Triton X-114 two-phase system was accomplished by determining its partition coefficient on the basis of enzyme activity. To that, concentration of COX activity in each phase has been determined by measuring enzyme activity and phase volumes after phase separation. The partition coefficient is defined as activity concentration in the rich phase over activity concentration in the depleted phase:\nK= [COX]rich / [COX]depleted\nResults are shown in Figure 5. Concerning the enzyme extracted from cells, partition coefficient reaches an optimum at 3% w/v detergent, decreasing at higher detergent concentrations. We do not have direct evidence to explain this decrease but a reason for it could be a change in the composition of the rich phase that may contain more components of the bacterial cell wall extracted at high detergent concentration, thus interfering in the partitioning of COX. In the case of the extracellular COX the higher the detergent concentration the larger the partition coefficient. As seen in table 4, the rich phase is quite more compact after phase separation in the culture broth than in the cell-extract and its relative volume is not proportional to the detergent concentration. Thus the rich phase is less hydrated, that is, more hydrophobic, with the culture broth than with the cell-extract and the more hydrophobic the higher the detergent concentration. As a result the partition coefficient for COX in the culture broth increases with the detergent concentration. Phase separation of Triton X-114 is affected by the presence of other surfactants such as Triton X-45 [28] and polyols such as glycerol [29], thus bacterial surfactants extracted during detergent treatment may affect phase separation as well. Natural surfactants may be also present in the broth but at a much lower concentration, therefore having less influence on the phase separation.\nFigure 5 Partition coefficient of cell-linked (•) and extracellular (•) COX in a Triton X-114 phase separation system.\nTable 4 Partitioning of cell-linked and extracellular COX after phase-separation of Triton X-114\nCOX total units COX activity (U/ml) K\n% Triton X-114 % volume of rich phase Rich phase Depleted phase Rich phase Depleted phase\nCell-linked\n1 10.8 8.5 234.8 78.7 263.2 0.30\n2 15.9 137.9 136.5 867.3 162.3 5.34\n3 29.6 276.5 87.1 934.1 123.7 7.55\n4 37.5 278.0 86.5 741.3 138.4 5.35\n6 41.8 280.0 85.0 669.8 146.0 4.58\nExtracellular\n1 6.4 5.0 113.7 78.2 121.5 0.64\n2 9.3 22.8 99.7 245.7 109.7 2.24\n3 10.7 58.8 69.4 550.5 77.7 7.08\n4 12.8 70.5 55.1 552.3 63.1 8.75\n6a 15.3 84.0 41 547.9 48.4 11.32\n6 15.3 390 41 2544 48.4 52.60\naCOX units in rich phase calculated as if no activation occurred: total units before partitioning (125) – units in depleted phase (41) The partitioning of commercial COX from Nocardia erythropolis and Pseudomonas sp. has been shown to depend on the detergent partitioning, since factors that affected the Triton X-114 distribution, such as temperature of partitioning, pH and phosphate concentration of the buffers, affected in the same way the distribution of enzyme [16]. Thus our results are in agreement with those findings. The reason for the different behavior of the detergent packing in cell extracts and in broth may lie in the fact that detergent partitioning is affected by physicochemical factors such as the presence of polyols, lipids, surfactants, etc [13], which can be present in cell extracts but not in the cell culture."}