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{"project":"2_test","denotations":[{"id":"19956594-3858868-88162829","span":{"begin":12100,"end":12102},"obj":"3858868"},{"id":"19956594-18826338-88162830","span":{"begin":12105,"end":12108},"obj":"18826338"},{"id":"19956594-1654141-88162831","span":{"begin":12306,"end":12308},"obj":"1654141"},{"id":"19956594-1654141-88162832","span":{"begin":12523,"end":12525},"obj":"1654141"},{"id":"19956594-15348163-88162833","span":{"begin":12722,"end":12724},"obj":"15348163"},{"id":"19956594-12059033-88162834","span":{"begin":12728,"end":12730},"obj":"12059033"},{"id":"19956594-12423600-88162835","span":{"begin":12865,"end":12867},"obj":"12423600"},{"id":"19956594-2501010-88162836","span":{"begin":13092,"end":13095},"obj":"2501010"},{"id":"19956594-14615161-88162837","span":{"begin":13098,"end":13101},"obj":"14615161"},{"id":"T83535","span":{"begin":12100,"end":12102},"obj":"3858868"},{"id":"T80520","span":{"begin":12105,"end":12108},"obj":"18826338"},{"id":"T19183","span":{"begin":12306,"end":12308},"obj":"1654141"},{"id":"T71084","span":{"begin":12523,"end":12525},"obj":"1654141"},{"id":"T41092","span":{"begin":12722,"end":12724},"obj":"15348163"},{"id":"T27516","span":{"begin":12728,"end":12730},"obj":"12059033"},{"id":"T49828","span":{"begin":12865,"end":12867},"obj":"12423600"},{"id":"T24507","span":{"begin":13092,"end":13095},"obj":"2501010"},{"id":"T95641","span":{"begin":13098,"end":13101},"obj":"14615161"}],"text":"Heat Treatment of Fetuin-A and Albumin as a Means to Induce Conformational Changes and to Trigger the Seeding of Minerals: Differences Seen between Metastable and Supersaturated Calcium and Phosphate Solutions\nThe seeding effects seen with both fetuin-A and albumin, manifested only in the presence of exogenously added calcium and phosphate, but not with metastable medium, were initially puzzling. The results would seem to indicate that the calcium and phosphate ions found in the DMEM may be stably complexed with other ionic and organic moieties, such that the added presence of fetuin-A and/or albumin is probably not sufficient to perturb this equilibrium. Viewed from a different perspective, it can also be inferred that the calcium (1.8 mM) and phosphate (0.9 mM) concentrations present in DMEM are not sufficient to saturate either fetuin-A or albumin, however little amount of either protein is added, such that the calcification-inhibitory potential associated with either one of these proteins might be overcome. Apparently, in the presence of additional calcium and phosphate ions added to the medium, there is now the binding of these excess free precipitating ions by these two proteins that, upon reaching saturation, may become the nuclei or nidi for the growth of apatite crystals.\nTo confirm this notion and to further correlate the results obtained for fetuin-A and albumin with those obtained earlier for whole serum with respect to nanoparticle formation, we next subjected both proteins to heat treatment for different lengths of time, following the same protocol used earlier with whole serum. We reasoned first that there is a fundamental difference seen with metastable and supersaturated calcium and phosphate solutions insofar as the mineral-seeding potential of fetuin-A and albumin is concerned that may be related to the conformational states of these protein molecules. We reasoned further that harsh treatments like boiling may induce the conformational changes required to unfold fetuin-A or albumin that in turn may allow either one or both to induce mineralization in a metastable medium like DMEM.\nIn Figure 7, both BSF and BSA were boiled for either 10, 30, or 120 min, followed by inoculation into DMEM in the absence (Fig. 7A) or presence (Fig. 7B) of exogenously added calcium and phosphate. Up to 2 mg/ml of BSF and 4 mg/ml of BSA were used for the experiments described here. As can be seen from Figure 7A, BSF that had been boiled for either 10, 30, or 120 min did not give any precipitation when inoculated into DMEM alone, in the absence of any added calcium or phosphate. In the same series of experiments, it could also be noticed that the addition of control, untreated BSF to DMEM also did not produce any precipitation (Fig. 7A, row labeled as “0 min Boiling Time”), in line with the earlier experiments that failed to detect any precipitation under these conditions (compare with the data shown in Figs. 4A and 5). On the other hand, pre-boiled BSA produced precipitation that could be readily detected following 1 month of incubation (Fig. 7A, “Boiled BSA” column). Precipitation appeared to be a function of the boiling time, with a 120-minute boiling treatment resulting in an immediate dose-dependent precipitation detectable on the first day following inoculation, presumably due to drastic protein denaturation. In fact, following this prolonged heat treatment (120 min), the BSA suspension became turbid and appeared to contain fine aggregates. After the first day, the turbidity of the inoculated medium did not seem to increase significantly when measured again after 1 month (Fig. 7A). On the other hand, boiling BSA for 10 or 30 minutes did not appear to create any turbidity increase, as evidenced by the turbidity measurements collected on the first day following inoculation (Fig. 7A). Turbidity however increased gradually over a period of 1 month, following a bell-shaped, dose-dependent curve, similar to what had been seen earlier with pre-boiled FBS (compare with Fig. 3A). That is, turbidity increased up to 0.4 mg/ml of BSA and decreased thereafter with higher protein concentrations. With both whole FBS and HS, we had also seen earlier evidence of protein denaturation with prolonged boiling for 120 minutes; a turbidity increase could also be seen readily on the first day following inoculation (Fig. 3A).\n10.1371/journal.pone.0008058.g007 Figure 7 Seeding of NB-like particles by boiled fetuin-A and albumin in metastable versus supersaturated medium.\n(A) Metastable medium: BSF and bovine serum albumin (BSA) were boiled for the time indicated on the left side. The boiled protein solutions were then diluted into DMEM, separately or together, to the final concentrations indicated on the top. A650 was then monitored following inoculation (“Day 1”) and after incubation in cell culture conditions for 1 month, as indicated on the right. Inoculation of either untreated BSF or BSF that had been boiled for either 10, 30, or 120 min did not result in any turbidity changes after 1 month of incubation. In contrast, pre-boiled BSA produced either bell-shaped or linear turbidity changes depending on the amount of boiling time. When added together, pre-boiled BSF and BSA produced additive effects. (B) Supersaturated medium: BSF and BSA were added to medium as in (A) that was also inoculated with 1 mM each of CaCl2 and NaH2PO4. Notice the various patterns of turbidity changes associated with the three protein combinations. See the text for further details. When inoculated together, BSF and BSA that had been pre-boiled for either 10 or 30 minutes no longer produced any precipitation even when observed after 1 month of incubation, suggesting in inhibitory influences exerted predominantly by BSF at the expense of BSA (Fig. 7A, “Boiled BSF+Boiled BSA” column). On the other hand, the addition of BSF and BSA that had been pre-boiled for 120 minutes also gave a dose-dependent increase in turbidity on the first day following incubation into DMEM, presumably due to the same insolubility of boiled BSA seen earlier. It should be further noted that the addition of both control and untreated BSF and BSA did not produce any turbidity change, in line with earlier results shown in Figures 4A and 5.\nTogether, these results indicate that BSF that had been boiled extensively retained its inhibitory effects on mineral precipitation in a metastable medium like DMEM, while pre-boiled BSA appeared to seed mineral growth. The seeding induced by pre-boiled BSA was replaced however by an inhibitory effect that was seen at higher BSA concentrations exceeding 1.2 mg/ml (results seen with BSA pre-boiled for 10 and 30 in). However, upon prolonged boiling for 120 minutes, BSA lost both its slow mineral-seeding as well as inhibitory potential.\nIn the presence of added calcium and phosphate, however, the following interesting observations were obtained (Fig. 7B). Firstly, with the addition of 1 mM calcium and phosphate alone, precipitation appeared in DMEM immediately and gradually increased for several days, after which it remained unchanged for up to 1 month (Fig. 7B, only “1 Month” reading shown). In fact, the readings obtained for “Day 4,” “Day 7,” and “1 Month” were virtually indistinguishable (data shown only for 1 month). The addition of control, untreated BSF produced a noticeable dose-dependent, inhibitory effect on this same precipitation that remained also unchanged for a period of observation of up to 1 month (Fig. 7B, row referred as “0 min Boiling Time”). Surprisingly, the inoculation of BSF that had been pre-boiled for 10 or 30 minutes resulted in a similar stable, dose-dependent inhibitory effect that also remained unchanged for at least 1 month. No additional precipitation that could be attributed to control or pre-boiled BSF was found. In contrast, BSF that had been pre-boiled for 120 minutes displayed markedly different behavior. Precipitation was enhanced in a dose-dependent manner by this boiled BSF that synergized with the precipitation produced by the addition of 1 mM calcium and phosphate. Apparently, the calcification-inhibitory effects attributed to BSF were at least partially removed by this extensive boiling.\nPre-boiled BSA produced marked different results. Firstly, both control, untreated BSA and BSA that had been pre-boiled for 10 or 30 minutes were shown to produce an immediate inhibitory effect on the precipitation mediated by 1 mM calcium and phosphate added to DMEM (Fig. 7B, see the “Boiled BSA” column in the “Day 1” panel). Precipitation then increased gradually over the course of the next 3 days (not shown). With additional incubation, the initial inhibitory effects seen associated with both untreated and pre-boiled BSA gave way to a marked dose-dependent increase that peaked at 1.2 mg/ml BSA (Fig. 7B). Pre-boiled BSA produced higher levels of turbidity as compared with control, untreated BSA (Fig. 7B). Still, in all 3 instances (untreated BSA, or BSA that had been pre-boiled for 10 or 30 min), a somewhat inhibitory effect was seen at a BSA concentration of 4 mg/ml. In contrast, BSA that had been pre-boiled for 120 minutes also showed an immediate precipitation on “Day 1” that synergized with the precipitation induced by 1 mM calcium and phosphate alone; this same precipitation did not increase thereafter (compare “Day 1” and “1 Month” panels in Fig. 7B), indicating that, as before, the turbidity seen associated with this form of boiled BSA most likely resulted from protein denaturation, and this denaturation may have removed both the seeding and inhibitory potentials of this protein.\nFinally, adding both pre-boiled BSF and BSA to supersaturated DMEM containing 1 mM calcium and phosphate produced the same additive inhibitory effects seen earlier with this type of protein mixture (Fig. 7B, compare with similar column in Fig. 7A). As before, it appears that the presence of BSF, be it untreated or pre-boiled, markedly reduced the seeding potential attributed to BSA alone.\nOur results indicate that, compared to albumin, fetuin-A is more resistant to boiling and that it is predominantly a calcification inhibitor even in the presence of exogenously added calcium and phosphate (e.g. in supersaturated calcium solutions). Thus, even upon prolonged incubation for at least 1 month, these same inhibitory effects seen with pre-boiled fetuin-A against supersaturated solutions remained intact and appeared to predominate over any seeding tendencies. Albumin, on the other hand, was shown here to be less inhibitory on mineralization and it also appeared to be more sensitive to boiling. Thus, boiled albumin seeded minerals spontaneously, in the absence of exogenously added calcium and phosphate. However, it is clear that both albumin and fetuin-A, upon being boiled up to 30 minutes, retained their respective mineralization-inhibitory capabilities, a property that was lost upon more extensive boiling (120 min), at which point, seeding prevailed.\nFrom our data, it can be further concluded that we have been able to induce calcium nanoparticle seeding in a metastable medium like DMEM with boiled albumin, but not with fetuin-A. It should be pointed out that our initial intention was to use boiling as a tool to unfold or denature both proteins so as to demonstrate their mineral seeding capabilities in the context of a metastable medium. However, in spite of repeated efforts to unfold or denature fetuin-A using boiling as well as other treatments detailed below, we have failed to induce mineral seeding with fetuin-A under metastable conditions.\nFor instance, we attempted to immobilize either albumin or fetuin-A, or both, onto various plastic substrates, including 24-well plates and 96-well ELISA plates made of polystyrene or polyvinylidine fluoride (PVDF) membranes. Earlier studies had shown that several proteins in solution tended to inhibit the precipitation of calcium phosphate whereas the same proteins promoted the nucleation of calcium phosphate minerals when they were adsorbed onto a solid substrate [97]–[100]. This dual effect was attributed to the possibility that the protein in solution could cover entirely the nascent minerals, thus masking their growing sites and thereby inhibiting crystal growth [99], [101]. On the other hand, adsorption onto a substrate would prevent the protein from covering the growing crystals, but would instead expose chemical groups needed for the formation of the first crystal nucleus [99], [101]. In the case of albumin, this protein was shown to induce collagen mineralization when present in solution at low concentrations whereas higher concentrations inhibited mineral formation [81], [82]. Albumin also promoted the mineralization of HAP when this mineral was coated with the protein and immersed in metastable solutions [83], [84]. In contrast to these studies, other reports have shown that albumin would essentially inhibit precipitation of calcium phosphate irrespective of whether it was adsorbed onto a substrate or remained free in solution [102]–[112]. From these observations, it thus remained unclear how these proteins would behave when immobilized under conditions known to favor the growth of the putative NB in culture.\nIn order to verify the effect of immobilized albumin and fetuin-A on the formation of mineral nanoparticles, we attempted to immobilize both proteins onto a hard surface with the intention of inducing the conformational changes required to induce mineral formation. Immobilization was performed by either drying the proteins onto the plates overnight, by incubating the protein solutions overnight at 4°C, or by using cross-linking reagents like poly-lysine or octadecyltrichlorosilane (OTS) to maximize the immobilization of the proteins on the plate. A wide range of protein concentrations ranging between 20 µg/ml to 40 mg/ml was used. Coating was ascertained by washing the plates subsequently with water or HEPES buffer and staining the plates for protein ( Materials and Methods ). The wells coated with proteins were put in contact with DMEM and incubated for several weeks in cell culture conditions, similar to the culture of NB. In spite of repeated attempts, none of these treatments resulted in precipitation of particles directly from DMEM, even after prolonged incubation up to 1 month and monitoring with both A650 readings and direct microscopic examination using phase-contrast microscopy (data not shown). From these observations, it appeared that the presence of fixed fetuin-A or albumin was insufficient to induce the formation of mineral nanoparticles in metastable solutions. Since albumin, but not fetuin-A, that had been boiled could indeed induce mineral formation in metastable DMEM, while neither one was able to induce mineralization when fixed onto solid substrates, it is unclear whether our negative results here are due to the particular experimental conditions used. More experiments are clearly needed before a definitive conclusion can be reached regarding the seeding capability of either protein, especially in the case of fetuin-A."}

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PMC:2779105 / 68977-84124 JSON TXT