Biomacromolecules 2005, 6, 1858-1863
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Structural Characterization of r-Terminal Group of Natural Rubber. 2. Decomposition of Branch-Points by Phospholipase and Chemical Treatments Lucksanaporn Tarachiwin,† Jitladda Sakdapipanich,*,†,‡ Koichi Ute,§ Tatsuki Kitayama,§ and Yasuyuki Tanaka† Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakhonphatom 73170, Thailand, and Department of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan Received January 26, 2005
The treatment of deproteinized natural rubber (DPNR) latex with phospholipases A2, B, C, and D decreased significantly the long-chain fatty acid ester contents in DPNR and also the molecular weight and Higgins’ k′ constant, except for phospholipase D treatment. This indicates the presence of phospholipid molecules in NR, which combine rubber molecules together. Transesterification of DPNR resulted in the decomposition of the functional group at the terminal chain-end (R-terminal), including phospholipids and formed linear rubber molecules. The addition of small amounts of ethanol into the DPNR solution reduced the molecular weight and shifted the molecular weight distribution (MWD) comparable to that of transesterified DPNR (TE-DPNR). The addition of diammonium hydrogen phosphate into DPNR-latex in order to remove Mg2+ ions yielded a slight decrease in molecular weight and a slight shift in MWD. The phospholipids are expected to link with mono- and diphosphate groups at the R-terminal by hydrogen bonding and/or ionic linkages. The decrease in the molecular weight and Huggins’ k′ constant of DPNR demonstrates the formation of linear molecules after decomposition of branch-points by this treatment, showing that phospholipids participate in the branching formation of NR. The branch-points formed at the R-terminus are postulated to originate predominantly by the association of phospholipids via micelle formation of long-chain fatty acid esters and hydrogen bonding between polar headgroups of phospholipids. Introduction Natural rubber (NR) from HeVea brasiliensis is presumed to be composed of two kinds of functional groups at both initiating- and terminating-ends, i.e., the ω- and R-termini, in which the former is bonded with proteins, whereas the latter is presumed to be linked with phospholipids.1 Both terminal groups originate branch-points and soft-gel in NR.1 The branch-points of gel in NR containing proteins are decomposed to form linear and branched molecules by a deproteinization process using a proteolytic enzyme with a surfactant.2 It has been further proved that the addition of small amounts of polar solvent into the rubber solution decomposes the hydrogen bonding between proteins associated with rubber molecule.1 Transesterification of deproteinized natural rubber (DPNR) in toluene solution with sodium methoxide decomposes the residual branch-points containing ester linkages.2 In the previous paper, we showed the presence of phospholipid molecules at the R-terminus of the rubber chain linked via mono- and diphosphate groups at
the chain-end.3 The treatment of DPNR with lipase and phosphatase decreased the content of long-chain fatty acid ester, molecular-weight, and Huggins’ k′ value. The decrease of molecular weight accompanied with the decrease of k′ value indicates that the R-terminal functional group participates in the branching formation in NR. In an attempt to gain greater understanding of the structure and role of phospholipids at the rubber chain-end, additional experiments were carried out to decompose the branch-points selectively by treatment of DPNR with specific activities for phospholipids, i.e., phospholipases, as well as chemical treatment. By a combination of these treatments with the results from molecular weight analysis by gel permeation chromatography (GPC) and dilute solution viscometry, the treatment with phospholipases A2, B, C, and D as well as chemical treatment of DPNR will provide confirmatory evidence illustrating the structure of the R-terminus of NR and the origin including the mechanism of branching formation in NR. Experimental Section
* Corresponding author. E-mail:
[email protected]. Tel: +66-2-8893116. Fax: +66-2-889-3116. † Department of Chemistry, Faculty of Science, Mahidol University. ‡ Institute of Science and Technology for Research and Development, Mahidol University. § Osaka University.
Freshly tapped natural rubber latex (FL-latex) was diluted to 30% dry rubber content (DRC) and subjected to deproteinization to remove protein layer covered on the rubber particles by incubation with 0.04% w/v protease (KP-3939,
10.1021/bm058004p CCC: $30.25 © 2005 American Chemical Society Published on Web 06/04/2005
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Decomposition of Branch-Points
Kao) in the presence of 0.5% w/v Triton X-100 at 37 °C for 12 h, followed by successive centrifugation twice at 20 300 g for 30 min and redispersed with 0.2% w/v Triton X-100 to make 10% DRC. The deproteinized rubber latex (DPNRlatex) was incubated at 37 °C for 3 days with phospholipase A2, B, and C obtained from Bovine pancreas (activity 5.7 units/ mg solid, Sigma) at pH 8, Vibrio species (activity 20 units/ mg protein, Sigma) at pH 8 and C. perfringe (C. Welchii, activity 5.9 units/mg solid, Sigma) at pH 7.3, respectively, and at 30 °C with phospholipase D obtained from peanut (activity 54 units/ mg solid, Sigma) at pH 5.6 for the appropriate phospholipase concentration. The resulting latex was centrifuged at 8680 g for 20 min and coagulated with methanol. The gel content of DPNR was determined to be zero. Free fatty acids or nonlinked fatty acids were removed by acetone extraction in a Soxhlet apparatus for 48 h. Acetone-extracted DPNR (AE-DPNR) was dissolved in toluene solution (AR-grade, LabScan) at a concentration of 1% w/v. Then, ethanol (AR-grade, LabScan) was added into the rubber solution at a concentration of 1% w/v and stirred at room temperature (∼25 °C) for 24 h. The solid rubber was obtained by precipitation of the rubber solution into methanol as mentioned above. The treatment of DPNR-latex with diammonium hydrogen phosphate (DAHP) was carried out by the addition of 5% w/v DAHP into DPNR-latex (20% DRC). The mixture was then incubated at room temperature (∼25 °C) with stirring for 1day followed by centrifugation at 6000 g for 1 h. The cream fraction was then re-dispersed in 0.5% Triton X-100 and recentrifuged at 6000g for 1 h. Dried rubber was obtained by coagulating the latex with methanol and dried in vacuo at 50 °C. Transesterification of the rubber from acetone extractedDPNR (AE-DPNR) was carried out in 0.5% w/v toluene by reaction with freshly prepared sodium methoxide and stirring at room temperature for 3 h. The resulting transesterifieddeproteinized natural rubber (TE-DPNR) was purified by precipitation of the rubber solution using excess of methanol and then dried in vacuo. The molecular weight of NR was determined by gel permeation chromatography (JASCO-Borwin) using two columns in series, packed with polystyrene-divinylbenzene copolymer gel. Tetrahydrofuran (LabScan, HPLC grade) was used as an eluent with a flow rate of 0.5 mL/min at 35 ( 0.01 °C, monitoring with a refractive index detector. Narrow molecular-weight distribution polyisoprenes (Polymer Standard Service GmbH, Germany) were used as standards. The samples for GPC measurement were prepared at a concentration of 0.05% w/v in THF and filtered through a Millipore prefilter and 0.45 µm membrane filter (Alltech). The intrinsic viscosity was measured with a single bulb Ubbelohde viscometer (SCHOTT, CT52) at 30 ( 0.02 °C using toluene as the solvent (analytical grade). The viscosity viscosity-average molecular-weight was calculated according to the Mark-Houwink’s equation as given in (1):4 [η] ) kM h av
(1)
Figure 1. Chemical structure of L-R-phosphatidylcholine and reaction site for phospholipase decomposition.
Here, k and a are constant values of 33.1 × 10-5 and 0.71, respectively. The Huggins’ k′ constant was calculated using eq 2: ηsp/c ) [η] + k′[η]2c
(2)
where ηsp/c, [η], c, M h v, and k′ represent the reduced viscosity, intrinsic viscosity, concentration expressed as g/dL, viscosityaverage molecular-weight, and Huggins’ constant, respectively. The content of long-chain fatty acid ester groups was determined by FTIR (FT/IR 460 Jasco). A series of mixtures of methyl stearate and synthetic cis-1,4-polyisoprene (Kuraprene IR10) was used to make a calibration curve for the FTIR analysis of ester groups in NR. The rubber samples for FTIR analysis were prepared by casting 1% w/v solutions of the rubber in chloroform on a KBr disk. The film was scanned with 100 scans at a resolution of 4 cm-1. The height ratio of peaks at 1739 cm-1 (CdO) to 1664 cm-1 (CdC) was plotted against the concentration of the ester groups in the range of 2-40 mmol/kg rubber. Results and Discussion The presence of acylglycerides, presumably as phospholipids at the R-terminus of the rubber chain, was confirmed by the structural change of the rubber chain after treatment with lipase and phosphatase as shown in the previous report.3 Confirmatory evidence for the presence of phospholipids at the R-terminus of the rubber chain can be provided by treatment of DPNR-latex with various phospholipases with different sites of reaction on the phospholipid. The reaction sites of phospholipids that can be hydrolyzed by phospholipases are shown in Figure 1. Phospholipase A2 hydrolyzes L-R-phosphatidylcholine (PC) at the C1 position to give L-Rlyso-phosphatidylcholine and fatty acids, whereas phospholipase B produces glycerol phosphorylcholine by decomposition of fatty acid ester groups at both C1 and C2. Watersoluble organic phosphates and choline are liberated by the treatment of PC with phospholipases C and D, respectively. The presence of phospholipids in NR was confirmed by the decrease in the content long-chain fatty acid ester groups together with the decrease of molecular weight and intrinsic viscosity, [η], after treating DPNR-latex with phospholipases A2, B, C, and D, as tabulated in Table 1. The fatty acid ester content of DPNR decreased significantly from 6.1 to 1.6, 2.2, and 3.0 mol per rubber molecule by treatment with
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Table 1. Characteristic of DPNR and Phospholipase-Treated DPNR with Phospholipases A2, B, C, and D concentration ratio of phospholipase:DPNR w/w A2
B
C
D
characteristic
AE-DPNRa
1:5
1:100
1:5
1:5
ester content (mol/rubber chain) M h n (× 105) M h w (× 105) M h w/M hn [η] M h v (× 105) k′
6.05
1.62
4.41
2.25
3.03
1.93 4.34 3.84 5.12 7.92 0.47
0.63 5.21 8.19
1.53 3.88 2.52 3.33 4.32 0.19
1.06 4.41 4.94 3.40 4.45 0.29
1.72 7.40 4.31 4.00 5.59 0.47
a
AE-DPNR: Acetone-extracted DPNR.
phospholipases A2, C, and D, respectively, at phospholipase concentration ratios of 1:5 against rubber weight. On the other hand, the treatment with phospholipase B showed a slight decrease to 4.4 mol per rubber molecule at a ratio of phospholipase:DPNR of 1:100 by weight. This might be due to the use of a smaller amount of phospholipase B during treatment of DPNR-latex. Ordinarily, it is difficult to expect a decrease of fatty acid ester content by the treatment of DPNR-latex with phospholipases C and D, because they hydrolyze only phosphate and choline linkages, respectively, in a phospholipid, and cannot decompose fatty acid ester linkages. The effect of phospholipases C and D on the decrease of fatty acid ester content can be interpreted by considering the solubility of phospholipids after treatment with phospholipases C and D. This could be explained by considering the fact that the hydrolysis of polar groups in the phospholipid by phospholipase C and D will increase the solubility of the resulting lipids in acetone during the purification process. The hydrolysis of polar groups could also decompose the branchpoints of DPNR formed by phospholipids at the R-terminus of the rubber molecule. The treatment of DPNR-latex with phospholipases A2, B, and C decreased significantly the M h n and [η] values to about one-third to one-half of the original values, whereas no change was observed in the case of treatment with phospholipase D. This result indicates that most parts of the branch-points originate from the long-chain fatty acid groups at C1, C2, and phosphate group at C3 in the phospholipid molecule. The mechanism of branching formation formed by phospholipids will be discussed afterward. It is noteworthy that the treatment of DPNR-latex with phospholipase D substantially decreased the ester content, whereas a slight decrease was observed in the molecular weight and [η] values. This suggests that the choline group in the phospholipid had no predominant role in the formation of branch points at the R-terminus of the rubber chain. Similarly, the Huggins’ k′ value of DPNR shown in Table 1 was higher than those of DPNR treated with phospholipases B and C, whereas that of DPNR treated with phospholipase D was almost the same as that of the original DPNR. Taking into account the decrease in the molecular weight and [η] as well as k′ values, it is clear that some parts of the branch-points of DPNR were decomposed by the treatment with phospho-
Figure 2. Molecular-weight distribution of acetone extracted DPNR before and after treatment with phospholipases.
lipases B and C, whereas phospholipase D could not decompose the branch-points. It is established that NR is composed of long-chain branched molecules.5,6 showing a bimodal MWD with high and low molecular-weight peaks from 1.0 × 106 to 2.5 × 106 and from 1.0 × 105 to 2.0 × 105, respectively.7,8 Figure 2 shows the molecular weight distribution (MWD) of DPNR before and after treatment with phospholipases A2, B, C, and D. No significant change was observed for the bimodal MWD of DPNR in the case of treatment with phospholipase D. On the other hand, the treatment with phospholipases A2, B, and C resulted in the apparent shift of the bimodal peak, showing the clear shift of the high molecular-weight peak to the low molecular-weight one and overlapped with the peak of low molecular-weight fraction. This apparent change in the MWD is presumed to be due to decomposition of branch-points to form linear molecules. These findings support strongly the assumption of the role of fatty acid ester and phosphate groups in the phospholipid molecule for the branching formation in DPNR. The morphology of lipid aggregates depends on the total phospholipid concentration, the ratio of the constituents, and the temperature.9,10 Most phospholipids, due to their amphiphilic nature, resist solubilization in polar solvents as well as nonpolar solvents by the formation of micelles and reverse micelles, respectively.11 It has been reported that the carbon atoms in fatty acid chains of dipalmitoyl lecithin form micelles in chloroform and bilayers in deuterium oxide.12 The long-chain acyl groups in triglycerides, which are extremely water insoluble, exhibit interfacial properties in water. Thus, they orient at air-water interfaces with the polar glyceryl portion interacting with water.13 In small unilamellar PC vesicles, triolein (TO) has a finite solubility and a preferred orientation with the carboxyl groups at the aqueous interface.14 A similar result was also observed for TO in multilamellar PC.15 The amide proton in phosphosphingolipid has been reported to be mostly involved in the intermolecular hydrogen bondings which link neighboring phospholipids through bridging water molecules. On the contrary, in the absence of water, the NH group participates in intramolecular hydrogen bonding that restricts the mobility of the phosphate group.16 Based on the characteristics of phospholipids, the formation of branch-points in DPNR has then been presumed to originate from the micelle formation of long-chain fatty acid
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Decomposition of Branch-Points
Table 2. Molecular Weight of (A) DPNR Precipitated from Toluene Solution after Treatment with and without 1% v/v Ethanol and Transesterifed DPNR and (B) DPNR Coagulated from Latex after Treated with 5% w/w Diammonium Hydrogen phosphate (DAHP) sample
M hn
M hw
M h w/M hn
(A) solution
AE-DPNR AE-DPNR + 1% ethanol TE-DPNR
1.80 × 105 1.01 × 105 1.04 × 105
1.33 × 106 5.64 × 105 4.02 × 105
7.41 5.57 3.88
(B) latex
DPNR DPNR + 5% DAHP
1.30 × 105 1.26 × 105
7.86 × 105 6.31 × 105
6.05 5.01
esters of phospholipids and hydrogen bonding with the phosphate group at the R-terminus of the rubber chain and the phospholipid polar headgroup. The branch-points formed by hydrogen bonding could be confirmed by the addition of small amounts of polar solvent into the rubber solution. The MWD of DPNR in toluene solution treated with ethanol is shown in Figure 3. AE-DPNR showed the bimodal MWD rich in the high molecular-weight fraction. By a combination with the analysis with GPC and dilute solution viscosity of NR, the high molecular-weight fraction was expected to form tri- or tetra-functional branch-points.5,6 The number of branch-points increased with increasing molecular-weight of the fractionated rubbers. By extrapolation of the plot between the number of branch-points and log molecular weight, it was postulated that the low molecular-weight rubber fraction consisted of linear molecules.5 We reported that transesterification of NR leads to the decomposition of branch-points resulting in a decrease in the molecular weight and a remarkable increase of the low molecular-weight fraction.1 It was presumed, therefore, that the increase in the low molecular-weight fraction in the bimodal MWD after transesterification is derived from the decomposition of branchpoints to form linear molecules. The addition of 1% v/v ethanol into DPNR solution led to the decrease in molecular-weight and narrower MWD comparable to TE-DPNR as shown in Table 2. It can be deduced that the addition of ethanol into DPNR solutions resulted in the decomposition of branch-points caused by hydrogen bonding between phospholipids to form linear molecules. Infrared spectroscopy has been used as a method of studying intermolecular interactions caused by hydrogen bonding, because the vibrational modes of the donor and acceptor groups are sensitive to this interaction leading to a change in a vibrational characteristic.17 The difference in
Figure 3. Molecular-weight distribution of acetone extracted DPNR, transesterified DPNR and DPNR precipitated from toluene containing 1% v/v ethanol.
vibrational frequencies can be used to observe direct or indirect hydration or dehydration effects, intra- or intermolecular binding of hydroxyl or amino groups, as well as cationmediated changes at the bilayer surface.18 Hydrogen bonding to the oxygen atoms leads to a weakening of the vibrational force constants and therefore to a decrease in the frequency. In addition, in sphingolipids or other lipids containing amide groups, the amide I band is sensitive to hydrogen bonding and to electrostatic interactions.18 It has been reported that the asymmetric O-P-O vibrational band is extremely sensitive to hydration, because of the shift of its frequency from 1250 for dry to 1230 cm-1 in a hydrated bilayer.19 Based on these studies, it is possible to confirm the formation of hydrogen bonding due to phospholipids in DPNR by FTIR spectroscopy. Low molecular-weight compounds in DPNR were obtained from the toluene/methanol fraction of precipitation from toluene solution into methanol. The toluene solution was treated with and without 1% v/v ethanol in order to check the effect of polar solvent. Figure 4 compares the FTIR spectra of low molecular-weight compounds with that of standard PC. As mentioned above, the CdO and O-P-O stretching bands provide an important clue to differentiate the phospholipid conformations. Normally, a phospholipid shows two CdO stretching bands centering at 1738-1742 and 1724-1729 cm-1. The presence of two CdO peaks is expected to be due to the different degree of hydration and/or hydrogen bonding to CdO20 The O-P-O asymmetric stretching of the hydrated phospholipid bilayer is usually observed at 1230 cm-1, whereas dried or anhydrous lipid always appears at 30 cm-1 higher frequency.21
Figure 4. FTIR spectra of low molecular-weight compounds extracted from DPNR by precipitation from (A) DPNR in toluene containing 1% v/v ethanol, (B) DPNR in toluene, and (C) L-R-phosphatidylcholine (PC).
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Figure 5. Molecular-weight distribution of DPNR from DPNR-latex treated with 5% w/w diammonium hydrogen phosphate (DAHP).
It can be seen in Figure 4 that the low molecular-weight compounds from DPNR and that of DPNR treated with 1% v/v ethanol in toluene give FTIR spectra similar to that obtained from PC. This indicates that phospholipids are a component of rubber molecules in terms of both linked and free molecules. It is remarkable that a new band was observed at 1713 cm-1 in both DPNR and DPNR treated with toluene containing 1% v/v ethanol. This small band clearly indicates the presence of hydrogen bonding between phospholipid molecules. However, the relative intensity of the 1713 cm-1 band in low molecular-weight compounds from DPNR treated with toluene containing 1% v/v ethanol was lower than that without ethanol. This indicates that hydrogen bonding between phospholipid molecules in NR can be decomposed by ethanol. The presence of hydrogen bonding between phospholipids was further confirmed by the O-P-O stretching band. The low molecular-weight compounds from DPNR showed the O-P-O asymmetric stretching at 1219 and 1240 cm-1, whereas the former stretching was absent in both PC and low molecular-weight compounds obtained from DPNR solution containing 1% v/v ethanol. This result strongly suggests that phospholipids in DPNR rubber aggregate or link together via hydrogen bonding through both phosphate and carboxyl groups. The result mentioned above demonstrates that branchpoints in DPNR originate partly by the aggregation of phospholipids and phosphate groups by hydrogen bonding. Here, it may be possible to consider another linkage to hold together two or more rubber chains at the R-terminus. It was found that the Mg2+ ion in latex acts to form branch-points during storage of FL-latex in the presence of ammonia.22 If the Mg2+ ion links the phosphate group at the R-terminus of DPNR and phospholipids, a single rubber chain should be composed of phospholipid and phosphate groups. It has been reported that the removal of Mg2+ ions resulted in a decrease of gel content and slowed the increase in gel content of long preserved FL-latex.23 Excess amounts of Mg2+ ions in FL-latex could be removed by the addition of 5% w/v diammonium hydrogen phosphate (DAHP).23 Thus, in this study, the effect of Mg2+ ions on the branching formation deriving from phospholipids was analyzed by the addition of 5% w/v DAHP into DPNR-latex. The MWD and molecular weight of DPNR-latex treated with DAHP are shown in Figure 5 and Table 2, respectively. The addition of DAHP into DPNR-latex caused only a slight decrease in
Tarachiwin et al.
Figure 6. Presumed structure for the R-terminal group of NR (a) terminated with monophosphate and (b) terminated with diphosphate.
Figure 7. Presumed structure of branch-points in NR originated by R-terminal functional group.
the molecular weight, although the MWD was comparable to that of DPNR. This indicates that Mg2+ ions have less effect on the branching formation than hydrogen-bonding. This supports the idea mentioned above that branching formation in DPNR is mainly caused by hydrogen bonding of phospholipids aggregated to phosphate groups at the R-termini of rubber molecules. Based on all of the above results, the branch-points are expected to originate mainly from micelle formation of the long-chain fatty acid ester groups and by the aggregation of phospholipids between polar headgroups via hydrogen bonding. In addition, the branch-points formed by ionic linkages, i.e., Mg2+ ions, between phosphate groups were also considered to be a part of branch-points. Incidentally, it may possible to postulate the structure of the R-terminal groups of NR as illustrated in Figure 6. The mono- and diphosphate groups at the R-termini were postulated to link to phospholipids, based on branching character, by hydrogen bonding or ionic linkages. Now, it is possible to propose the structure of branch-points in DPNR based on the findings in the present work as shown in Figure 7. The branch-points are due to aggregation of the phospholipids which are linked to phosphate or diphosphate groups at the R-termini. The phospholipids are aggregated together predominantly by hydrogen bonding between phospholipid polar headgroups, i.e., phosphate or carbonyl groups and partially by ionic linkages.
Decomposition of Branch-Points
Conclusion Phospholipids are one of the constituents associated with the R-termini of rubber chain consisting of mono- or diphosphate groups. The decrease of molecular weight and k′ values after treatment with phospholipases B and C indicate that long-chain fatty acid esters and phosphate groups in phospholipid participate in branching formation in DPNR. The branch-points in DPNR are postulated to be due to phospholipids at the R-terminus, in which most of the branch-points originate from the micelle formation of the long-chain fatty acid ester groups and hydrogen bonding between polar headgroups, i.e., phosphate groups of phospholipids. The branching formation formed by ionic linkage, i.e., Mg2+ ions, between polar headgroups were also considered to be an inferior effect. Acknowledgment. Financial supports from the Thailand Research Fund PHD/0142/2543 and RSA4680009. References and Notes (1) Tangpakdee, J.; Tanaka, Y. Characterization of Sol and Gel in HeVea Natural Rubber. Rubber Chem. Technol. 1997, 70, 707. (2) Tangpakdee, J.; Tanaka, Y. Purification of natural rubber. J. Nat. Rubber Res. 1997, 12, 112. (3) Tarachiwin, L.; Sakdapipanich, J.; Ute, K.; Kitayama, T.; Bamba, T.; Fukusaki, E.; Kobayashi, A.; Tanaka, Y. Structural Characterization of R-terminal Group of Natural Rubber. 1. Decomposition of Branch-Points by Lipase and Phosphatase Treatments. Biomacromolecules 2005, 6, 1851. (4) Subramaniam, A. Gel Permeation Chromatography of Natural Rubber. Rubber Chem. Technol. 1972, 45, 346. (5) Angulo-Sanchez, J. L.; Caballero-Mata, P. Long chain branching in natural HeVea rubber - determination by gel permeation chromatography. Rubber Chem. Technol. 1981, 54, 34. (6) Fuller, K. N. G.; Fulton, W. S. The influence of molecular weight distribution and branching on the relaxation behavior of un-crosslinked natural rubber. Polymer 1990, 31, 609. (7) Bristow, G. M.; George, M.; Westall, B. The molecular weight distribution of natural rubber. Polymer 1967, 8, 609. (8) Westall, B. The molecular weight distribution of natural rubber latex. Polymer 1968, 9, 243.
Biomacromolecules, Vol. 6, No. 4, 2005 1863 (9) Gabriel, N. E.; Roberts, M. F. Spontaneous formation of stable unilamellar vesicles. Biochemistry 1984, 23, 4011. (10) Sanders, C. R.; Hare, B. J.; Howard, K. P.; Prestegard, J. H. Magnetically oriented phospholipid micelles as a tool for the study of membrane associated molecules. Prog. NMR Spectrosc. 1994, 26, 421. (11) Murari, R.; Abd. El-Rahman, M. M. A.; Wedmid, Y.; Parthasarathy, S.; Baumann, W. J. Carbon-13 Nuclear Magnetic Resonance Spectroscopy of Phospholipids in Solution. Spectral and stereochemical Assignments Based on 13C-31P and 13C-14N Couplings. J. Org. Chem. 1982, 47, 2158. (12) Metcalfe, J. C.; Birdsall, N. J. M.; Feeney, J.; Lee, A. G.; Levine, Y. K.; Partington, P. Carbon-13 NMR spectra of lecithin vesicles and erythrocyte membrane. Nature 1971, 233, 199. (13) Smaby, J. M.; Brockman, H. L. Regulation of cholesteryl oleoate and triolein miscibility in monolayers and bilayers. J. Biol. Chem. 1987, 262, 8206. (14) Hamilton, J. A.; Small, D. M. Solubilization and localization of triolein in phosphotidylcholine bilayers: A Carbon-13 NMR study. Proc. Natl. Acad. Sci., U.S.A. 1981, 78, 6878. (15) Gorrissen, H.; Tulloch, A. P.; Cushley, R. J. Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers. Chem. Phys. Lipids 1982, 31, 245. (16) Stacey, R.; Ferguson-Yankey, Borchman, D.; Grant Taylor, K.; Donald B. DuPre´, and Cecilia Yappert, M. Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin. Biochim. Biophys. Acta 2000, 1467, 307. (17) Hu¨bner, W.; Blume, A. Interactions of lipid-water interface. Chem. Phys. Lipids 1998, 96, 99. (18) Blume, A. Properties of lipid vesicles: FT-IR spectroscopy and fluorescent probe studies. Curr. Opin. Colloid Interface Sci. 1996, 1, 64. (19) Fringeli, U. P.; Gu¨nthard, H. H. Infrared membrane spectroscopy. In Membrane Spectroscopy; Grell, E., Ed.; Springer: Berlin, 1981; pp 270. (20) Blume, A.; Hu¨bner, W.; Messner, G. Fourier Transform Infrared Spectroscopy of 13CdO-Labeled Phospholipids. Hydrogen Bonding to Carbonyl Groups. Biochemistry 1988, 27, 8239. (21) Wong, P. T. T.; Mantsch, H. H. High-pressure infrared spectroscopic evidence of water binding sites in 1,2-diacylphospholipids. Chem. Phys. Lipids 1988, 46, 213. (22) Tarachiwin, L.; Sakdapipanich, J.; Tanaka, Y. Gel formation in natural rubber latex 2. Effect of Magnesium Ion. Rubber Chem. Technol. 2003, 76, 1185. (23) Tarachiwin, L.; Sakdapipanich, J.; Tanaka, Y. Gel formation in natural rubber latex 1. Effect of Additives. Rubber Chem. Technol. 2003, 76, 1175.
BM058004P
