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价值工程1概述机舱罩作为风力发电机组的重要部件，是风力发电机组的防护结构，使风力发电机组能在恶劣的气象环境中正常工作，保护内部设备和人员不受风、雨、雪、盐雾、紫外辐射等外部环境因素的侵害。
在这种环境条件下，要保证风电机组正常工作20年，就要求机舱罩具有高质量、高可靠性。
2玻璃钢材料玻璃钢具有质量轻、强度高、耐化学腐蚀、电绝缘、透微波等许多优良性能，而且成型方法简单，可以一次成型各种大型或具有复杂构型的制品[1]。
聚酯玻璃钢和其他材料的拉伸强度与弹性模量等性能对比见表1[1]。
从表1中数据可以看出，聚酯玻璃钢的比强度高于型钢、硬铝和杉木，但比模量较低。
经过合理的结构设计，可以弥补其弹性模量的不足，而且充分发挥其比强度以及其他优良性能[1]。
故综合考虑到机舱罩的性能要求选择玻璃钢作为合适的材料，制造机舱罩的玻璃钢是由不饱和聚酯树脂和玻璃纤维增强材料构成的。
———————————————————————收稿日期：2012年10月16日。
作者简介：王凯（1985-），男，陕西榆林人，现供职许昌许继风电科技有限公司，中级工程师，毕业于西安理工大学，本科学历，从事风电机组机舱罩开发工作。
风力发电机组机舱罩制造简述The Manufacture Outline on Nacelle Housing of Wind Turbine Generator王凯WANG Kai ；史航SHI Hang ；程林志CHENG Lin-zhi ；刘二恩LIU Er-en（许昌许继风电科技有限公司，许昌461000）（Xuchang Xuji Wind Power Technology Co.，Ltd.，Xuchang 461000，China ）摘要：机舱罩作为风力发电机组保护壳体，其可靠性决定了风电机组运行的稳定性和使用寿命。
本文结合玻璃钢材料、成型工艺、模具要求、尺寸控制、质量缺陷等多方面内容对风力发电机组机舱罩的制造过程进行简述。
BD Biosciences Pharmingen United States 877.232.8995Canada 888.259.0187Europe 32.53.720.211Japan 0120.8555.90Asia Pacific 65.6861.0633Latin America/Caribbean 55.11.5185.9995For country-specific contact information, visit /how_to_order/Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD Biosciences will not be held responsible for patent infringement or other violations that may occur with the use of our products. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express BD Pharmingen Technical Data SheetANNEXIN V-PE APOPTOSIS DETECTION KIT IPRODUCT INFORMATIONCatalog Number:559763Components:51-65875X :Contents:Annexin V-PE100 tests; buffered in 50 mM Tris (pH 8.0) with 80 mM NaCl, 0.2%BSA, 1 mM EDTA, 0.09% (w/v) sodium azide.51-68981E :Contents:7-AAD (7-Amino-actinomycin D)2.0 ml in PBS and 0.09% sodium azide and FBS Wash Buffer.51-66121E :Contents:Annexin V Binding Buffer, 10X Concentrate 50 ml solutionBACKGROUNDApoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis.The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca 2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS (reviewed in 1). Annexin V may be conjugated to fluorochromes such as Phycoerythrin (PE). This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis.2-5Since externalization of PS occurs in the earlier stages of apoptosis, Annexin V-PE staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. Annexin V-PE staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V-PE is typically used in conjunction with a vital dye such as 7-Amino-actinomycin (7-AAD, Cat. No. 68981E) to allow the investigator to identify early apoptotic cells (Annexin V-PE positive, 7-AAD negative).2-5 For example, cells that are viable are Annexin V-PE and 7-AAD negative; cells that are in early apoptosis are Annexin V-PE positive and 7-AAD negative; and cells that are in late apoptosis or already dead are both Annexin V-PE and 7-AAD positive.2-5 This assay does not distinguish, per se, between cells that have already undergone apoptotic death and those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both Annexin-PE and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Annexin V-PE and 7-AAD negative (viable, or no measurable apoptosis), to Annexin V-PE positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Annexin V-PE and 7-AAD positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both Annexin V-PE and 7-AAD positive,in of itself, reveals less information about the process by which the cells underwent their demise.SPECIFICITY AND PREPARATIONA n n e x i n V -PE i s a s e n s i t i v e pr o be for i d e n t i f yi n g apoptoti c ce l l s .2-5 It bi n d s to n e g a ti v e l y ch a r g e d ph o s p h o l i pi d sur f a c e s (K d of ~5 x 1 0 -2)6w i t h a hi g h e r s p eci f i c i t y for ph o s p h a t i d y l s e r i n e (PS) th a n m o st othe r ph o s p hol i p id s . De f i n e d cal c i u m a n d s a l t con c en t r a ti o n s a r e r e q u ir e d for A n n e xi n V-PE bi n d i n g a s d e s c r i b e d i n th e An n e x in V -PE Stai n i n g Pr o tocol. Puri f i e d r e c om b i n a n t An n e x in V wa s con j u ga t e d to PE un d e r opti m u m con d i t i o n s . An n e x in V -PE i s r o uti n e l y te s t e d usi n g pr i m a r y cel l s or ce l l l i n e s in d u ced to un d e r g o an a p optotic d e a th .NOTE: Methods for utilizing Annexin V binding on adherent cells (i.e., monolayer) have been described by van Engeland et al 8 and Casciola-Rosen etal 7. However, these methods are not routinely tested for Annexin V-PE. AnnexinV-FITC Microscopy kit (Cat.No. 550911) is recommended for detection of Annexin V binding to adherent cells.USAGE AND STORAGEA p pli c a t ion s in c l u d e flow cytom e tr y (5 µl /tes t ). Se e th e A n n e xi n V-PE Stai n i n g Pr o tocol for us a g e i n for m a t ion . Stor e A n n e xi n V-PE at 4 ° C .(p l e a se see n e x t p a g e )BD Biosciences Pharmingen United States 877.232.8995Canada 888.259.0187Europe 32.53.720.211Japan 0120.8555.90Asia Pacific 65.6861.0633Latin America/Caribbean 55.11.5185.9995For country-specific contact information, visit /how_to_order/Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD Biosciences will not be held responsible for patent infringement or other violations that may occur with the use of our products. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express REFERENCES1.Raynal, P. and H.B. Pollard. 1994. Annexins: The problem of assessing the biological role for a gene family of multifunctional calcium and phospholipid-binding proteins. Biochemica et Biophysica Acta. 1197:63-93.2.Vermes, I., C. Haanen, H. Steffens-Nakken, and C. Reutelingsperger. 1995. A novel assay for apoptosis. Flow cytometric detection ofphosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Meth. 184:39-51.3.Martin, S.J., C.P. Reutelingsperger, A.J. McGahon, J.A. Rader, R.C. van Schie, D.M. LaFace, and D.R. Green. 1995. Early redistribution of plasmamembrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J.Exp. Med. 182:1545-1556.4.Koopman, G., C.P. Reutelingsperger, G.A. Kuijten, R.M. Keehnen, S.T. Pals, and M.H. van Oers. 1994. Annexin V for flow cytometric detection ofphosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415-1420.5. Homburg, C.H., M. de Haas, A.E. von dem Borne, A.J. Verhoeven, C.P. Reutelingsperger and D. Roos. 1995. Human neutrophils lose their surfaceFc γRIII and acquire Annexin V binding sites during apoptosis in vitro . Blood 85:532-540.6. Andree, H.A., C.P. Reutelingsperger, R. Hauptmann, H.C. Hemker, W.T. Hermens and G.M. Willems. 1990. Binding of vascular anticoagulant α(VAC α) to planar phospholipid-binding proteins. J. Biol. Chem. 265:4923-4928.7. Casciola-Rosen, L., A. Rosen, M. Petri, and M. Schlissel. 1996. Surface blebs on apoptotic cells are sites of enhanced procoagulantactivity:Implications for coagulation events and antigenic spread in systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA 93:1624-1629.8. van Engeland, M., F.C. Ramaekers, B. Schutte, and C.P. Reutelingsperger. 1996. A novel assay to measure loss of plasma membrane asymmetryduring apoptosis of adherent cells in culture. Cytometry 24:131-139.ANNEXIN V -PE STAINING PROTOCOLAnnexin V -PE is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. It relies on the property of cells to lose membrane asymmetry in the early phases of apoptosis. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external environment.Annexin V is a Ca 2+-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying apoptotic cells with exposed PS. 7-Amino-actinomycin (7-AAD) is a standard flow cytometric viability probe and is used to distinguish viable from nonviable cells. Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. Cells that stain positive for Annexin V-PE and negative for 7-AAD are undergoing apoptosis. Cells that stain positive for both Annexin V-PE and 7-AAD are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both Annexin V-PE and 7-AAD are alive and not undergoing measurable apoptosis.Reagents1.Annexin V-PE (Cat. No. 51-65875X). Use 5 µl per test.2.7-AAD (Cat. No. 51-68981E). Use 5 µl per test. 7-AAD (7-Amino-actinomycin D) is a convenient, ready-to-use solution of the nucleicacid dye that can be used for the exclusion of nonviable cells in flow cytometric assays. 7-AAD fluorescence is detected in the far red range of the spectrum (650 nm long-pass filter).1,23. 1 0 X A n n e x i n V B i nd i n g B u f fe r . (Ca t . N o . 5 1 -66 1 2 1 E ). 0.1 M H e p e s /N a O H (pH 7 .4 ) 1 .4 M N a Cl , 25 m M Ca C l 2.3Th e s o luti o n w a s 0 .2 µm s t e r i l e fil t e r e d . For a w o r k i n g s o l u tion (1X ), di l u te 1 pa r t bi n d i n g buffe r to 9 par t s di s t i l l e d H 20. T h i s w i l l y i e l d a wor k i n g s o luti o n of 1 0 m M H e p e s /N a O H (pH 7 .4 ) 1 4 0 m M Na C l , 2 .5 mM Ca C l 2. Stor e both th e 1 0 X con c e n tr a t e a n d w o r k i n g sol u tion a t 2 - 8 ° C .Staining1.Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 106 cells/ml.2.Transfer 100 µl of the solution (1 x 105 cells) to a 5 ml culture tube.3.Add 5 µl of Annexin V -PE and 5 µl of 7-AAD.4.Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.5.Add 400 µl of 1X binding buffer to each tube. Analyze by flow cytometry within one hour.For Research Use Only. Not For Diagnostic or Therapeutic Use.Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD PharMingen will not be held responsible for patent infringement or other violations that may occur with the use of our products.Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD Biosciences will not be held responsible for patent infringement or other violations that may occur with the use of our products. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express 10010110210310M1M2100Annexin V-PE7-A 10110210310410010110101010010110210310M1M2B T reated100Annexin V-PE7-A A D101102103104100101102103104Annexin V-PE: A tool for identifying cells that are undergoing apoptosis.T cells were left untreated (A) or treated for 4 hr with 4 µM Camptothecin (B). Cells were incubated with Annexin V -PE in a buffer containing 7-Amino-actinomycin (751-68981E) and analyzed by flow cytometry. Untreated primarily Annexin V -PE and 7-AAD negative, indicating that they were viable and SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRYThe following controls are used to set up compensation and quadrants 1.Unstained cells.2.Cells stained with Annexin V -PE alone (no 7-AAD).3.Cells stained with 7-AAD alone (no Annexin V-PE).Other Staining ControlsA cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with Annexin V-PE and with both Annexin V-PE and 7-AAD. It is important to note that the basal level of apoptosis and necrosis varies considerably within a population. Thus, even in the absence of induced apoptosis, most cell populations will contain at least a minor percentage of cells that are positive for apoptosis (Annexin V-PE positive, 7-AAD negative or Annexin V-PE and 7-AAD positive).The untreated population is used to define the basal level of apoptotic and dead cells. The percentage of cells that have been induced to undergo apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated population from percentage of apoptotic cells in the treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged membrane and stain positive for 7-AAD as well as for Annexin V-PE. Thus the assay does not distinguish between cells that have already undergone an apoptotic cell death and those that have died as a result of necrotic pathway, because in either case the dead cells will stain with both Annexin V-PE and 7-AAD.INDUCTION OF APOPTOSIS BY CAMPTOTHECINAnnexin V-PE。
High Resolution, Real Time Studies of Protein-Lipid Interactions Using Hemilayer Membrane MimicsIntroductionDual Polarisation Interferometry (DPI) is an important enabling tool for cell biology, particularly the study of membrane proteins, their behaviour and interactions (1). This application note describes the use of DPI for the real time, quantitative analysis of protein-lipid interactions implicated in the formation of lamellipodia structures involved in cell migration. The molecular system under investigation involved phospholipids commonly found in cell membranes and known to be involved in the regulation of a range of membrane proteins, PtdIns(4,5)P2(D-myo-phosphatidylinositol 4,5-biphosphate known as PIP2) and PtdIns(3,4,5)P3 (D-myo-phosphatidylinositol 3,4,5-triphosphate known as PIP3) and their interactions with WAVE2, a WASP family verprolin homologous protein.Cell migration is one of the ways that cells respond to signals from the extracellular environment. Receptor molecules on the plasma membrane sense the location and intensity of extracellular signals and determine the appropriate direction for cell movement. Recent studies have revealed the important roles played by membrane lipids and the cytoskeleton in cell migration. It is clear that cells recruit the PIP3 at the leading edge to establish cell polarity in response to chemo-attractant gradients. However, the mechanism of how this polarity is followed by formation of the leading edge is still obscure. The protein WAVE2 is expressed ubiquitously in various tissues, and several researchers have reported that localization of WAVE2 at the leading edge is crucial for lamellipodium formation. However, the molecular mechanisms underlying this localization are poorly understood.In this study, a membrane mimic formed by self-assembly of solubilised phospholipid deposited on a C18 surface was used to support the PIP molecules under investigation. Subsequently, we investigated the affinity of interaction between the protein WAVE2 and both PIP2 and PIP3 supported in this phospholipid hemilayer, in an attempt to confirm which PIP binds preferentially to WAVE2.ExperimentalThe DPI experiments were performed on a Farfield Ana Light®instrument. The surface used was a C-18 functionalised silicon oxynitride Ana Chip™. The temperature of the samples was controlled throughout to 20o C. All buffers and reagents were analytical grade or higher, and solutions were degassed prior to use.Lipid Hemilayer Membrane Mimic Formation:The phospholipid vesicles used in these experiments contained PC:PtdIns:PIP2 or PIP3 at 48:48:4 (mole ratio) and were prepared by extrusion through a 0.1µm cyclopore filter. PIP2 and PIP3 vesicles were coated on the surface of different channels of the C-18 Ana Chip™. The lipids were bound in a monolayer onto C-18 tips, forming the lipid bilayer-like structure composed of C-18 carbon chains and lipids. The buffer used was 10mM HEPES, 150mM NaCl, 3mM EDTA, pH7.5.WAVE2 Protein – PIP Lipid Interaction Studies: Studies of the interactions between WAVE2 and PIP2 and PIP3 were performed by immobilizing vesicles containing PIP2 and PIP3 on different instrument channels as above, and introducing WAVE2 to both simultaneously. It is known that the pleckstrin homology (PH) domain of phospholipaseCδ1 (PLCδ1) shows a strong affinity for PIP2, and the PH domain of Akt interacted specifically with PIP2 and PIP3, and therefore these were included as controls. All proteins were injected at different concentrations in the same buffer (association phase). The Ana Chip™ was regenerated by injection of 10mM NaOH. Bindings were examined at four or more different protein concentrations, and then K D, k ass, and k diss values were calculated from curve fitting over the initial 60 seconds of association.Results and DiscussionWAVE2 Protein – PIP Lipid Interaction Studies:Figure 1illustrates the binding curves for the proteins on the respective instrument channels containing PIP2 and PIP3. The corresponding k ass, k diss and K D values are tabulated in Figure 2. The K D was calculated from curve fitting and the errors for each point from the fitted curve were within ±0.01% and ± 0.03%, respectively. It is concluded that WAVE2 binds preferentially to PIP3 rather than to PIP2 in vitro, supporting the view that recruitment of WAVE2 by PIP3 is an essential process for lamellipodia formation at the leading edge.Figure 1: Association curves for PIP3 (top) and PIP2 (bottom) with WAVE2, also showing positive (AktPH) andnegative (PLCδ1PH) controlFigure 2 k ass, k diss and K D values calculated from the binding curves showing WAVE2 has a much higheraffinity for PIP3 vesicles than for PIP2 vesicles(*At these concentrations PLCd1PH binding to PIP3 is so weak affinity constants were not calculated)Conclusions and BenefitsThese studies demonstrate DPI as an enabling technique for the formation of phospholipid membrane mimics and the subsequent study of the interactions of proteins with their phospholipid-binding partners. A simple self-assembled hemilayer can be formed by deposition of phosphlipids in liposome buffer or by the rupture of vesicles on a C-18 surface. Kinetics of interaction can be determined directly from the binding responses.These experiments show DPI can be applied to the study of membrane protein systems. The Ana Light® instruments and their experimental protocols give the researcher a unique combination of high-resolution data in real time on thickness, refractive index (density) and surface coverage from a bench top technique. The Ana Light® is an important enabling tool for protein biochemists giving them the ability to:•Clearly understanding the molecular mechanisms involved in protein-lipid interactions•Produce and validate viable phospholipid membrane mimics for use in membrane protein studies•Obtain real time assurance of supporting lipid integrity in such studies•Generate high-quality affinity parameter data for macromolecular interactions to help in the understanding of the molecular mechanisms underlying cellular processes•Avoid the limitations and ambiguities that are inherent in other established techniques for such studies, and provide the final results and analysis rapidlyReferences(1)Tsukasa Oikawa, Hideki Yamaguchi, Toshiki Itoh, Masayoshi Kato, Takeshi Ijuin, Daisuke Yamazaki, Shiro Suetsugu and Tadaomi Takenawa; PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia, Nature Cell Biology, 6, 420-428 (2004)Farfield gratefully acknowledges that these experiments were carried out in Professor Tadaomi Takenawa’s laboratory in the Department of Biochemistry, University of Tokyo and are reproduced here with his kindpermission.For further applications information contact: applications@ orTelephone the applications team on +44 (0) 870 950 9717。
NOVEMBER 2013 | VOL. 12 NO. 11 | TAPPI JOURNAL 19Dissolving pulp is the raw material for manufactur-ing rayon and many other cellulose derivatives. As an important quality parameter, reactivity (sometimes referred to as accessibility or processability) is used to represent the reactivity of dissolving pulp toward carbon disulfide in the downstream processing plants. More spe-cifically, it is used to assess the reactivity of the hydroxyl groups on glucose units of cellulose chains toward car-bon disulfide [1,2]. The increased reactivity of dissolving pulp implies improvements in the quality of cellulose products, and may lower the required dosage of carbon disulfide [3,4], an expensive and toxic chemical in the rayon industry.Several methods have been developed to determine the reactivity of dissolving pulp. The most popular one, the Fock test, was reported in 1959 [5]. In this method, the dissolving pulp sample is mixed with sodium hydroxide and excess amounts of carbon disulfide to form a solution of cellulose xanthate. Cellulose is regenerated from this solution in the presence of sulfuric acid, and its quantity is determined to obtain the percentage of reacted cellulose (known as the Fock reactivity). Evidently, the Fock test is a simplified viscose pro-cess on a laboratory scale [6,7]. Compared with other meth-ods, such as iodine sorption [8], water retention value [9], phosphitylation and quantitative 31P nuclear magnetic reso-nance (NMR) spectroscopy [10], and viscose filter value (which is commonly used in the viscose rayon industry, but complex and requires special equipment), the Fock test is easy to handle under conventional laboratory conditions [7,11]. The Fock test generally has been accepted in assessing thereactivity of dissolving pulps produced from various raw ma-terials by either acid sulfite or pre-hydrolysis kraft processes [10,12].However, Fock test results have not been consistent. In a study by Christoffersson [13], the Fock reactivity results con-ducted in the same laboratory under the same conditions, at the same time, varied from 56.9% to 64.7%, and the standard deviation was 13% for parallel tests conducted at different times (Fock reactivity results varied from 33.3% to 57.6%).Improvement in the Fock test for determining the reactivityof dissolving pulpCHAO TIAN, LINQIANG ZHENG, QINGXIAN MIAO, CHRIS NASH,CHUNYU CAO, and YONGHAO NIDISSOLVING PULPPEER-REVIEWEDABSTRACT: The Fock test is widely used for assessing the reactivity of dissolving pulp. The objective of thisstudy was to modify the method to improve the repeatability of the test. Various parameters that affect the repeat-ability of the Fock test were investigated. The results showed that Fock reactivity is dependent on testing conditions affecting the xanthation between cellulose and carbon disulfide, such as the moisture content of the pulp sample, sodium hydroxide (NaOH) concentration, xanthation temperature, carbon disulfide dosage, and xanthation time. The repeatability of the test was significantly improved using the following modified testing procedure: air dried sample in the constant temperature/humidity room, xanthation temperature of 66°F (19°C) in a water bath, xanthation time of 3 h, NaOH concentration of 9% (w/w), and 1.3 mL carbon disulfide.Application: The improved Fock test can give good repeatability in the reactivity test of dissolving pulp inresearch and development (R&D) laboratories and production lines.1. The poor repeatability of the Fock test performed on the same dissolving pulp sample but at different dates. Conditions: Tests were conducted according to the method described by Kvarnlöf (2007) and Östberg et al. [15].DISSOLVING PULP20 TAPPI JOURNAL | VOL. 12 NO. 11 | NOVEMBER 2013This poor repeatability was also seen in our study (Fig. 1), which was carried out on the same sample, following the same procedure, but on different days. This is no doubt unac-ceptable for both research work and quality control in the production line.Accordingly, the objectives of this study are to identify the reasons for the poor repeatability of the Fock test and to de-velop an improved testing procedure that will provide ac-ceptably repeatable results. To accomplish those objectives, the testing conditions affecting the xanthation of cellulose, such as the sodium hydroxide (NaOH) concentration, xantha-tion temperature, carbon disulfide dosage, and xanthation time, were systematically studied. Then, an improved Fock test procedure, which gave acceptable repeatability, was pro-posed and verified.EXPERIMENTALDetermination of Fock reactivityTable I lists characteristics of the three commercial dissolv-ing pulp samples provided for this study by different mills in Canada. For xanthation and regeneration of cellulose, 0.50 g of pulp sample (calculated as oven dry [o.d.] pulp) was weighed and placed in a 250 mL Erlenmeyer flask with a stop-per. After 50 mL NaOH solution was added, this flask was placed in a shaker, submerged in a water bath, and shaken for 10 min. As soon as the carbon disulfide was added, the flask was sealed with plastic paraffin film and continued to be shaken at 250 rpm for several hours at the specified tempera-ture. When the time was up, deionized water was immedi-ately added to the flask to give the solution a total weight of 100 g. After a vigorous shake, the solution was transferred to a tube with stopper and centrifuged at 5000 rpm for 15 min to separate dissolved cellulose from unreacted cellulose. Then, 10 mL of the supernatant was pipetted into a 100 mL flask and neutralized with 3 mL sulfuric acid of 20% (w/w). The cellulose was regenerated for 15-20 h and degassed to re-move carbon disulfide (CS 2).For measurement of the dissolved cellulose, 20 mL sulfuric acid (H 2SO 4) (68% w/w) was added to the 100 mL flask and the suspension was shaken for 1 h to acidify the regenerated cellulose. Thereafter, a 10 mL potassium dichromate (K 2Cr 2O 7) solution (1/6 M) was added to the flask and the mixture was reboiled for 1 h, during which the oxidation took place. Whenthe mixture was cooled to room temperature, it was diluted into a 100 mL volumetric flask and 40 mL of this solution was transferred into a 250 mL Erlenmeyer flask. As soon as 5 mL potassium iodide (10% w/w) was added, the solution was ti-trated with sodium thiosulfate (0.1 N) using starch as the in-dicator. The volume of consumed sodium thiosulfate was re-corded to obtain the percentage of cellulose that reacted with carbon disulfide, according to the following Eq. (1):Dissolved cellulose (%) =(1)where, M is the molecule weight of glucose unit (162 g/mol), m is the o.d. weight of the pulp sample (g), v 1 is the volume of K 2Cr 2O 7 added (0.01 L), c 1 is the concentration of K 2Cr 2O 7 solution (1/6 M), v 2 is the volume of consumed Na 2S 2O 3 (L), c 2 is the concentration of Na 2S 2O 3 solution (0.1 N), () means 40 mL diluted sample is taken out from 100 mL volumetric flask and titrated, () means each dichromate ion consumes six thiosulfate ions, () means each glucose unit consumes four dichromate ions, and () means 10 mL sample (equal to 10.4 g) is taken out from the 100 g viscose liquid.RESULTS AND DISCUSSIONEffect of testing conditionsThe NaOH concentration plays an important role in viscose rayon production. Before xanthation, dissolving pulp is first mercerized using a 10%-20% NaOH solution in an indepen-dent step to form alkali cellulose [16-18], which then reacts with carbon disulfide [13,15]. For the Fock test, the merceriza-tion, the xanthation, and the dissolution of cellulose are car-ried out in the same step. In the literature, various NaOH con-centrations were adopted by different researchers, including 8% w/w [19], 9% w/w [15,20], and 90 g/L [6,11]. To determine the effect of the NaOH concentration on the Fock reactivity, Fock tests on two pulp samples with various NaOH concentra-tions were conducted; Fig. 2 shows the results.Evidently, the Fock reactivity of dissolving pulp is very sensitive to the NaOH concentration in the xanthation step, especially in the range of 8%-10%. Based on these results, the NaOH concentration needs to be controlled precisely to min-imize the testing error, and a specified NaOH concentration must be given for the Fock test to compare the results from different researchers.Cellulose mercerization is essential for xanthation, by which the cellulose is swollen and alkali cellulose (Na-cellu-lose) is formed. The swelling degree of cellulose or transition from cellulose to Na-cellulose is dependent on the concentra-tion of NaOH. At low NaOH concentrations, usually below 10%, the cellulose swelling or transition of cellulose increases significantly with increase in the NaOH concentration; at high NaOH concentrations, the transition to Na-cellulose will reachI. Characteristics of dissolving pulp samples used in this study.DISSOVING PULPNOVEMBER 2013 | VOL. 12 NO. 11 | TAPPI JOURNAL 21the plateau, but the swelling of cellulose may decline [1,21]. Any processing conditions that can introduce variation in the NaOH concentration during the xanthation process should be minimized. Variation in the moisture content of the pulp samples, for example, can result in testing errors. To illustrate the effect of moisture brought in from the pulp sam-ple, a set of experiments were conducted on the same pulp sample (sample B), but with different moisture contents (Table II ).The results in Table II support the conclusion that the Fock reactivity of a pulp sample with lower moisture content is sig-nificantly higher than that of the same pulp sample with high-er moisture content. In fact, it can be calculated that under the conditions studied, for the air dried sample (moisture con-tent of 7.5%) and the sample with 73.66% moisture content, the additional moisture led to a 0.23% decrease in the NaOH concentration, which is then responsible for the much lower Fock reactivity for the 73.66% sample. Figure 2 also shows that at an NaOH concentration of 8%-10%, a small change in the NaOH concentration can cause a big change in the Fock reac-tivity. For this reason, pulp samples for the Fock test should be conditioned in the constant temperature/humidity room for 24 h, allowing a constant moisture content to be obtained.Xanthation temperatureIn the study by Fock in 1959 and most of the related studies by other researchers [4,6,7,11], the xanthation step in the Fock test was performed at room temperature. Because room tem-perature may fluctuate significantly, it can cause inconsisten-cy in the testing results. Figure 3 shows that the Fock reac-tivity differed from 58.50% to 16.45% when xanthation temperature was varied from 55°F to 93°F (13°C to 34°C).Several reasons might be responsible for the significant decrease in the Fock reactivity as the temperature increases: 1) the boiling point of carbon disulfide is only 115.3°F (46.3°C) and, therefore, more carbon disulfide loss might occur at a higher temperature through evaporation; 2) sodium hydrox-ide can swell cellulose to a higher degree at lower temperature [16,21-23], which facilitates the diffusion of carbon disulfide inside the cellulose structure; 3) xanthation is an exothermic reaction [16]; and 4) in the xanthation system, more byprod-ucts will be produced when the xanthation temperature in-creases, which competes for the consumption of carbon di-sulfide with the xanthation reaction [16,24-26].Similar results were reported by other researchers, though with various purposes. In a study by Rassolov and Finger [28], a sharp decrease in the maximum degree of esterification of cellulose at high xanthation temperature was observed. Ste-panova et al. [25] found that a higher xanthation temperature would lead to an increase in the formation rate of sulfur-con-taining by-products and simultaneously reduce the amount of carbon disulfide consumed in the xanthation of cellulose.Carbon disulfide dosageBased on the stoichiometry of Eq. (2), one can calculate that about 0.6 g of CS 2 would be required for xanthation of 0.5 g dissolving pulp, with the assumption that all of the three hy-droxyls on the glucose unit are xanthated (about 50%-70% ofthe hydroxyls can be practically xanthated [16,27]).2. Effect of sodium hydroxide (NaOH) concentration on the Fock reactivity. Conditions: Air-dried pulp sample of 7.5% moisture, 66°F (19°C) xanthation temperature, 1.3 mL carbon disulfide (CS2), 3 h xanthation time.3. Effect of xanthation temperature on Fock reactivity. Conditions: Air-dried sample A of 7.5% moisture, 9% w/w sodium hydroxide (NaOH) concentration, 1.3 mL carbon disulfide (CS 2), 3 h xanthation time.Sample Status Moisture Content ofSample (%)Fock Reactivity(%)II. Effect of the moisture content of pulp sample on Fock reactivity.DISSOLVING PULP22 TAPPI JOURNAL | VOL. 12 NO. 11 | NOVEMBER 2013(2)Thus, 1 mL of CS 2 (equal to 1.26 g), suggested in the Fock test published in 1959, seems to be in great excess for the Fock reactivity test. More recently, however, the dosage of CS 2 in the xanthation step was increased to 1.3 mL [6,7,11], while others still used 1 mL of CS 2 [14,15,20]. Figure 4 shows the effect of CS 2 dosage on Fock reactivity.At 75°F (24°C), the Fock reactivity reached a plateau at the CS 2 dosage of 1.3 mL (Fig. 4), and a lower CS 2 dosage than 1.3 mL would yield a lower reactivity; at 57°F (14°C), 1 mL of CS 2 would reach the plateau. Moreover, a much higher Fock reac-tivity was obtained at 57°F (14°C) than at 75°F (24°C), which is consistent with the results in Fig. 3.Xanthation timeThe xanthation time in the Fock test was not always the same in the previous studies. For example, the xanthation time was 3 h and the CS 2 dosage was 1 mL [14,15,20], while in others, 4 h of xanthation time and 1.3 mL of CS 2 dosage were used [6,7,11]. As shown in Fig. 5, the xanthation time has a strong impact on Fock reactivity, especially in the first 3 h (1 mL CS 2) or 4 h (1.3 mL CS 2). The two sets of conditions (1 mL CS2 for 3 h versus 1.3 mL CS 2 for 4 h) also yielded different reactivity results.Additionally, the xanthation time for the Fock reactivity to reach a plateau is much longer than the suggested 3-4 h. It was 5 h when 1 mL CS 2 was used, but no plateau was reached, even after 6 h of xanthation time when 1.3 mL CS 2 was used. For commercial viscose production, however, the xanthation time is usually 1-2 h [16,29].The conditions for cellulose xanthation in the Fock test are very different from those for practical viscose production. The dosage of CS 2 in the Fock test is about 10 times that used in viscose production (30%-35% of the α-cellulose weight) [16], which can lead to a higher xanthation degree of csellulose [27]. In addition, in viscose production, the diffusion of CS 2 and the xanthation of cellulose are more efficient than in the Fock test, because most of the excess NaOH solution is pressed out after mercerization and the CS 2 concentration rises to a high level in a short time [16,27]. For this reason, the xanthation time is much longer in the Fock test than in viscose production.Taking all factors into consideration, a 3 h xanthation time is recommended for the Fock test.Fock test repeatability with modified testing procedure Based on previous investigations, the following conditionswere proposed for the Fock test:4. Effect of carbon disulfide (CS 2) dosage on Fock reactivity. Conditions: Air-dried sample A of 7.5% moisture, 9% w/w NaOH concentration, 3 h xanthation time.5. Effect of Xanthation time on the Fock reactivity. Conditions: Air-dried sample A of 7.5% moisture, 9% w/w sodium hydroxide (NaOH) concentration, 66°F (19°C) xanthation temperature.III. The repeatability of the Fock test with the modified testing procedure.DISSOVING PULPNOVEMBER 2013 | VOL. 12 NO. 11 | TAPPI JOURNAL 231) T he pulp sample is air dried in a constant temperature/ humidity room. 2) T he xanthation is conducted in a water bath at 66°F (19°C).3) The dosage of CS 2 is 1.3 mL.4) The xanthation time is 3 h.5) The NaOH concentration is 9% (w/w).By following these procedures, samples A and B were sub-jected to the Fock test on different days. Table III shows the results. The standard deviation of these trials was about 1% (Table III), which is much lower than that shown in Fig. 1.These results supported the conclusion that the repeatability of the Fock test is significantly improved with the modified procedure.CONCLUSION This study addressed the repeatability of the Fock test. Thexanthation temperature of the Fock test is the most critical factor in the procedure. Room temperature was specified in the Fock test, but the actual temperature can vary significant-ly between different days, causing poor repeatability of themethod. Other factors, such as the NaOH concentration, CS 2 dosage, and xanthation time, can also affect the Fock test re-sults. By air drying the pulp sample in a constant temperature and humidity room, controlling the xanthation temperature at 66°F (19°C) in a water bath, using an NaOH concentration of 9% w/w, and using the xanthation time of 3 h and CS 2 dos-age of 1.3 mL, the modified Fock test can give good repeat-ability (the standard deviation of parallel tests performed in different days was about 1%). TJLITERATURE CITED1. Sixta, H., Handbook of Pulp , Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim, Germany, 2006.2. Strunk, P., “Charcterization of cellulose pulps and the influcence oftheir properties on the process and production of viscose and cel-lulose ethers,” Doctoral thesis, Umeå University, Umeå, Sweden,2012.3. Köpcke, V., “Improvement on cellulose acessibility and reactiv-ity of different wood pulps,” Licentiate thesis, Royal Institute of Technology, Stockholm, Sweden, 2008.4. Ibarra, D., Köpcke, V., Larsson, P. T., et al., Bioresour. Technol.101(19): 7416(2010).5. Fock, V.W., Das Papier 13(3): 92(1959).6. Engström, A.C., Ek, M., and Henriksson, G., Biomacromolecules 7(6):2027(2006).Cao NiNash Zheng Tian MiaoDISSOLVING PULP24 TAPPI JOURNAL | VOL. 12 NO. 11 | NOVEMBER 20137. Köpcke, V., “Conversion of wood and non-wood paper-grade pulpsto dissolving-grade pulps,” Doctoral thesis, Royal Institute of Technology, Stockholm, Sweden, 2010.8. Nelson, M.L., Rousselle, M.A., Cangemi, S.J., et al., Textile Res. J.40(10): 872(1970).9. Hopner, T., Jayme, G., and Urich, J.C., Papier 9(19/20): 476(1955).10. Filpponen, I. and Argyropoulos, D.S., Ind. Eng. Chem. Res. 47(22):8906(2008).11. Christoffersson, K.E., Sjöström, M., Edlund, U., et al., Cellulose 9(2):159(2002).12. Roffael, E., Holzforschung 42(2): 135(1988).13. Christoffersson, K.E., “Dissolving pulp- multivariate characterisa-tion and analysis of reactivity and spectroscopic properties,” Doctoral thesis, Umeå University, Umeå, Sweden, 2005.14. Kvarnlöf, N., “Activation of dissolving pulps prior to viscose prepa-ration,” Doctoral thesis, Karlstad University, Karlstad, Sweden, 2007.15. Östberg, L., Håkansson, H., and Germgård, U., BioResources 7(1):743(2012).16. Lin, Q., Chemical Fiber Technology , China Financial and EconomicPublishing House, Beijing, China, 1965.17. Irklei, V.M. and Goikhman, A.S., Fibre Chem. 20(1): 1(1988). 18. Gemci, R., Sci. Res. Essays 5(6): 560(2010).19. El-Ghany, N.A.A., Cellulose Chem. Technol. 46(1-2): 137(2012).20. Javed, M.A. and Germgård, U., BioResources 6(3): 2581(2011).21. Klemm, D., Philipp, B., Heinze, T., et al., Eds., ComprehensiveCellulose Chemistry , WILEY-VCH Verlag,Weinheim, Germany, 1998, Vol. 1, pp. 56-58.22. Richter, G.A. and Glidden, K.E., Ind. Eng. Chem. 32(4): 480(1940).23. Bergh, M., “Absorbent cellulose based fibers: Investigation ofcarboxylation and sulfonation of cellulose,” M.S. thesis, Chalmers University of Technology, Göteborg, Sweden, 2011.24. Stepanova, G.A., Grishchenko, V.I., Pakshver, A.B., et al., FibreChem. 12(1): 44(1980). 25. Stepanova, G.A., Pakshver, A.B., and Kaller, A.L., Fibre Chem. 14(1):27(1982). 26. Woodings, C. (Ed.), Regenerated Cellulose Fibers , WoodheadPublishing Limited, Cambridge, England, 2001, p. 47.27. Kotek, R., in Handbook of Fiber Chemistry (M. Lewin, Ed.) 3rd edn.,Taylor & Francis Group, Boca Raton, FL, USA, 2007, Chap. 10.28. Rassolov, O.P. and Finger, G.G., Fibre Chem. 13(4): 238(1981).29. Kuzicheva, N.A., Filicheva, T.B., Fedotova, V.K., et al., Fibre Chem .21(1): 22(1989).30. Xu, F., 人造纤维 (Artifical Fiber) 34(4): 4(2004).。
Reactivity of a Phospholipid Monolayer Model under Periodic Boundary Conditions:A Density Functional Theory Study of the Schiff Base Formation between Phosphatidylethanolamine and AcetaldehydeChristian Solı´s-Calero,Joaquı´n Ortega-Castro,and Francisco Mun˜oz*Institut d’In V estigacio´en Cie`ncies de la Salut(IUNICS),Departament de Quı´mica,Uni V ersitat de les IllesBalears,E-07122Palma de Mallorca,SpainRecei V ed:September16,2010;Re V ised Manuscript Recei V ed:October27,2010A mechanism for the formation of the Schiff base between an acetaldehyde and an amine-phospholipidmonolayer model based on Dmol3/density functional theory calculations under periodic boundary conditionswas constructed.This is thefirst time such a system has been modeled to examine its chemical reactivity atthis computation level.Each unit cell contains two phospholipid molecules,one acetaldehyde molecule,andnine water molecules.One of the amine-phospholipid molecules in the cell possesses a neutral amino groupthat is used to model the nucleophilic attack on the carboxyl group of acetaldehyde,whereas the other has acharged amino group acting as a proton donor.The nine water molecules form a hydrogen bond networkalong the polar heads of the phospholipids that facilitates very fast proton conduction at the ingperiodic boundary conditions afforded proton transfer between different cells.The reaction takes place intwo steps,namely,(1)formation of a carbinolamine and(2)its dehydration to the Schiff base.The carbinolamineis the primary reaction intermediate,and dehydration is the rate-determining step of the process,consistentwith available experimental evidence for similar reactions.On the basis of the results,the cell membranesurface environment may boost phospholipid glycation via a neighboring catalyst effect.IntroductionIn vivo nonenzymatic glycation is the covalent binding of a simple reducing sugar to a primary amino group in a biomol-ecule.The initial steps of this reaction are reversible and involve the addition of a primary amino group to a carbonyl group in glucose to give an intermediate carbinolamine that loses one molecule of water to produce an imine or Schiff base,the subsequent rearrangement of which leads to an Amadori product. Whereas nonenzymatic glycation in proteins and nucleic acids is very well documented,1,2lipid glycation has received com-paratively little attention.Some studies have shown that phosphatidylethanolamine(PE)reacts with glucose to form a PE-linked Amadori product(Amadori-PE)via an unstable Schiff base.3Amadori-PEs trigger oxidative modification in lipids via superoxides,promote vascular disease through their angiogenic action on endothelial cells,and may be involved in the development of diabetes by effect of aminophospholipids such as PE and phosphatidylserine being prone to nonenzymatic glycation under hyperglycemic conditions.4,5No inhibitor of lipid glycation has as yet been identified despite the potential biomedical significance of Amadori-PEs, most probably because of the lack of an effective lipid glycation model for inhibitor screening.6Thefirst step in PE glycation involves the formation of a Schiff base.Various aldehydes and ketones including glucose and acetaldehyde derived from ethanol metabolism have been experimentally found to form Schiff bases with PE.7-11Therefore,developing an appropriate lipid glycation model requires an accurate knowledge of the Schiff-PE forma-tion mechanism with provision for the reaction environment (specifically,a cell membrane and a solvated surface).On the basis of experimental evidence,lipid glycation is faster than protein glycation.12This can be ascribed to the chemical nature of membrane surfaces.Thus,the interfacial region of a membrane is known to establish electrostatic,hydrophobic,and/ or hydrogen-bonding interactions with various types of small molecules.13-15As a result,some functional groups in membrane surfaces may efficiently enhance lipid glycation via a neighbor-ing catalyst effect;also,solvated membrane surfaces may provide a favorable environment and lead to a faster reaction. Previous density functional theory(DFT)studies by our group allowed Schiff base formation mechanisms for vitamin B6 analogues to be elucidated;16,17also,the reactions of sugars and glycation target models with pyridoxamine have been the subject of various studies.18,19One of the most essential features of phospholipid molecules is their amphiphilic nature,with polar heads and nonpolar tails of fatty acid chains.Phospholipid bilayers are components of biological membranes and play an important role in biological systems.In general,zwitterionic lipids like phosphatidylcholine(PC)and PE have been more widely studied than anionic lipids.Molecular computer simulation is a powerful tool for studying the physical and chemical properties of phospholipids layers.A number of key factors in membranes including phase behavior,20,21stability and mechanical properties,22-26structural parameters,24,27and interactions with other molecules26,28,29have been investigated.DFT methods with periodic boundary conditions(PBCs)30,31 have been widely used in the study of the electronic structure of metal oxide surfaces,32-34zeolites35-37and other inorganic surfaces,38-42metal-organic frameworks(MOFs,metal oxide nodes linked by organic molecules),43,44self-assembled organic monolayers(SAMs),45,46and the interactions of these systems with small inorganic and organic molecules.*To whom correspondence should be addressed.Tel:+34971173252.Fax:+34971173426.E-mail:dqufmi0@uib.es.J.Phys.Chem.B2010,114,15879–158851587910.1021/jp1088367 2010American Chemical SocietyPublished on Web11/15/2010On the side of theoretical study of biological systems,Vener et al.47used DFT methods with PBCs to describe quantitatively the backbone -backbone and the side chain -backbone interac-tions in Ala-based secondary structures.Ferrari et al.48used the same methodology to study the helicoidal conformations of polyglycine chains.There are only few studies using DFT methods to phospho-lipids systems.Eriksson and co-workers 49studied the photo-oxidation of lipids by singlet oxygen.Recently,Snyder and Madura 50modeled the interaction between the phospholipids headgroup and the silica particles.Additionally,Sugimori et al.51and Krishnamurt et al.52studied the conformational space of some phospholipids molecules.To the best of our knowledge,no systematic theoretical studies about the chemical reactivity of phospholipid monolayers or bilayers has been carried out.In this work,which was intended to fill the existing gap,we used DFT level computations and PBCs for the first time to model a portion of the biological membrane surface with a view to investigating its reactivity.We elucidated the influence of a cell membrane solvated surface environment on the Schiff base formation reaction via an H-atom transfer mechanism.Overall,this theoretical study has enabled us to identify the key factors that make lipid glycation faster than other similar reactions.MethodologyA molecular model for 1,2-diacetoyl-D -L -phosphatidyletha-nolamine (DAPE)monolayer under PBCs was constructed.As can be seen in Figure 1,each cell in the model contained two DAPEs,acetaldehyde,and nine water molecules in a hydrogen bond network along the polar heads of the phospholipids.This model was used to study the Schiff base formation between DAPE and one molecule with a free aldehyde group.One of the DAPE molecules in the cell contained a neutral amino group intended to facilitate modeling of the nucleophilic attack on the carbonyl group of acetaldehyde,whereas the other DAPE possessed a charged amino group to assist its action as a proton donor or acceptor in some reactions steps.The purpose of including nine water molecules in the molecular model was not exclusively to simulate a water solvation environment;rather,the water molecules were intended to act as reactive species facilitating the process in the modeled membrane surface.PBCs have proved essential with a view to modeling phospholipid layer surfaces with a portion of the water solvation shell replicating enclosed molecules in the cell by rigid translation in the three Cartesian directions.This not only allowed atoms in the cell to interact with their images in nearby cells but also facilitated examination of the reactivity of phospholipid heads in the membrane surface model via DFT level calculations,which are computationally affordable.In addition,it allowed the passage of protons from one cell to another whenever proton transfer through a hydrogen bond network of water molecules was required in some step of the modeled reaction.All calculations were done with DMol3in MS Modeling 4.4,developed by Accelrys,Inc.53The initial,reactant model,and those for stationary points generated during formation of the Schiff Base,were developed in Materials Visualizer and initially optimized with electron DFT calculations using DMol3code.54,55All calculations were based on double numerical with polariza-tion (DNP)basis sets 54and the Perdew -Burke -Ernzerhof (PBE)generalized gradient approximation exchange-correlation functional.56It has been shown that the basis set superposition error (BSSE)in DNP is less than those in the Gaussian 6-311+G(3df,2pd)basis set and that extension of basissetFigure 1.Section of four unit cells of the initial model for two PE molecules,acetaldehyde,and the water hydrogen bond network.The atomsbelonging to one representative cell are represented by balls and sticks.Phosphorus and reactive atoms are labeled,and dotted lines represent hydrogen bonds.15880J.Phys.Chem.B,Vol.114,No.48,2010Solı´s-Calero et al.larger than DNP is unnecessary as long as geometrical properties and binding energies are discussed.57The basis sets are further characterized by the element dependent cutoff radii,which were chosen at thefine cutoff setting in DMol3.For the C,H,O,N, and P atoms,these values are3.7,3.1,3.3,3.4,and4.2Å, respectively.All computations were done with the maximum number of numerical integration mesh points available in DMol3,the density matrix convergence threshold being set to 10-6and the smearing parameter to0.005Ha.Accurate processing of hydrogen bonds was important here, and DMol3PBE calculations are known to provide highly accurate reaction enthalpies for molecules in the gas phase.58 The accuracy of DMol3in describing hydrogen bond strengths was previously tested in a theoretical study using settings similar to ours,which revealed consistency between calculated and experimental values.59The energetically most stable structures were identified by using the conjugated gradient algorithm.Transition states(TSs) were located by using the LST/QST method60and optimized via eigenvectors;to this end,the Newton-Raphson method was used to search for an energy maximum along a previously selected normal mode and a minimum along all other nodes. One TS per reaction was thus located,and harmonic vibration frequencies were calculated at the same level to ensure that every TS would have a single imaginary frequency.Results and DiscussionThe structures found allowed a detailed chemical pathway for the formation of a Schiff base between an acetaldehyde and a neutral amino group in a phospholipid(DAPE)to be established.Scheme1shows the atoms directly involved in the reaction and the overall process.As can be seen,the process essentially involves two steps,namely,carbinolamine formation (structures1-7)and dehydration to the Schiff base(structures 7-11).Table1lists the relative energies and∆G(1atm and 298K)values for each structure involved in the process,and Figure2shows the free energy profile.Carbinolamine Formation.The starting point for this stepwise process is structure E1(Scheme1),where the incoming amino group(N3)of the phospholipid is the agent of the nucleophilic attack on the carbonyl carbon in acetaldehyde(C1). The PBCs allow O2,O8,and O20to form hydrogen bonds with atoms in neighboring cells.The amine approach occurs in such a way that the O2-C1-N3angle is97.8°(see the relevant geometrical data for all structures in the Supporting Information) and starts at an N3-C1distance of2.704Å(Figure1).The gas phase activation barrier for direct addition of the amino group to the carbonyl group for other carbinolamine formation reactions is relatively high;61,62by contrast,that for our reaction is comparatively low(2.5kcal/mol).This can be ascribed to the presence of an explicit solvent that forms hydrogen bonds with the reactants and products alike,thereby facilitating addition of the amino group to the carbonyl carbon.Structure E3is in fact the zwitterionic form of the carbino-lamine.Atom O2,which is negatively charged,interacts with two water molecules via hydrogen bonds that are 1.647 (O2-H14)and1.644Ålong(O2-H9)(Figure3a).The former is a member of a hydrogen bond chain of three water molecules that facilities protonation of the charged oxygen(O2),thefinal proton donor being a charged amino group in another phos-pholipid chain.Proton transfer takes place via TS4,with a very low energy barrier(3.32kcal/mol).The PBCs allow proton H14 to cleave its bond to O13and be transferred from one face of the unit cell to the opposite face to bond to O2(Scheme1and Figure3).The resulting conformation in turn facilitates other proton transfers,the proposed pathway for proton migration along the modeled amine-phospholipid surface being that with the lowest energy among all potential pathways examined here. There is an alternative mechanism occurring in other systems where carbon-nitrogen bond formation and protonation of the oxygen atom occur in a concerted manner.16,17Also,there is experimental evidence that both pathways may occur concomi-tantly in acid-catalyzed O-methyloxime formation63and that the prevalence of one above the other is dictated by the stability of the zwitterionic form relative to the TS for the“concerted”pathway.The stability of the zwitterionic form increases,to an extent that is quantitatively predictable,with increasing in p K a for the parent amine.In our system,the presence of a membrane surface environment with negatively charged neighboring phosphate groups may stabilize the zwitterionic form E3,thereby leading to a prevalence of the above-described concerted mechanism for the acid-catalyzed formation of the carbinolamine. Intermediate E5can form a neutral carbinolamine by depro-tonation of N3.The most likely target for proton transfer from the amine is N6in a neighboring amino group.However,the distance from any of the hydrogen atoms in both atoms,5.335Å,is too long to allow direct transfer.This is the point where, again,three solvation water molecules play a reactant role.The hydrogen transfer from N3to N6takes place via a concerted TS(TS6in Scheme1)where the three water molecules exchange one of their hydrogen atoms to facilitate the proton transfer from N3in the incoming amino group and simultaneous release to N6,thereby restoring the original neighbor charged amino group and yielding the carbinolamine intermediate E7 once the free energy gap of3.99kcal/mol for this TS(TS6)is overcome(Figure4a).This kind of proton migration,known as the“Grotthuss mechanism”,has previously been reported for cell membrane surfaces and involves the chemical exchange of hydrogen nuclei between hydrogen-bonded surface water molecules and subse-quent rearrangement of the hydrogen bond network.64Besides, this mechanism allows a proton to diffuse throughout an entire hydrogen bond network of water at a rate considerably greater than that of conventional diffusion.65Water has been shown to take part in similar reactions in other simple systems where the energy barrier for carbinolamine forma-tion by proton transfer was found to be reduced if explicit water molecules were used to facilitate proton transfer.66On the basis of experimental work on other molecular systems,these protonation reactions are pH-dependent in acid-base equilibria.67,68 Dehydration.The next step in the reaction is dehydration of the carbinolamine to the corresponding Schiff base,which involves the concerted release of the O2-H14hydroxyl group and the transfer of one hydrogen from the protonated amino group in the second phospholipid chain through a water molecule to give the leaving water molecule and the protonated form of thefinal Schiff base,E9(Scheme1and Figure4).This step additionally causes the formation of an imine double bond between C1and N3,the distance between which is thereby reduced from1.470(E7)to1.322Å(E9)(Table2in the Supporting Information).As in other molecular systems,16,17,69 carbinolamine dehydration is the rate-determining step in the formation of the Schiff base,with an energy barrier of18.88 kcal/mol.E9contains a hydrogen bond between O2in the leaving water molecule and H19in another water molecule(Figure4),as well as a new hydrogen bond chain that facilitates the next reaction step,namely,formation of the corresponding neutral form ofReactivity of a Phospholipid Monolayer Model J.Phys.Chem.B,Vol.114,No.48,20101588115882J.Phys.Chem.B,Vol.114,No.48,2010Solı´s-Calero et al. SCHEME1:Mechanism of Schiff Base Formation between a PE Monolayer and Acetaldehyde,Using PBCs athe Schiff base (E11).The reaction involves deprotonation of N3in intermediate E9,aims at N6in the neutral amino group of the second phospholipid,and restores the initial,chargedamino group while giving the uncharged imine group C1d N3.Because the distance between the two groups is too long to allow direct transfer,four water molecules networked by hydrogen bonds act as a bridge to facilitate the passage of protons through a concerted TS (TS10in Scheme 1).This proton transfer is also subject to a small barrier (4.64kcal mol -1,Table 1and Figure 2).As compared with TS4and TS6,where the distance for all donor and acceptor hydrogen bond atoms involved in the protonation reaction is 2.4-2.6Å,and the reactions are subject to a very low energy barrier in both directions,some distances in TS10are longer (e.g.,2.807Åfor N3-O22distance and 2.785Åfor N6-O8).On the basis of our Schiff base formation model,water plays a prominent role in all proton transfers,where it acts as a bridge along which protons are transferred through water molecules networked by hydrogen bonds.Moreover,the whole reaction mechanism is governed to a great extent by the network of hydrogen bonds in the different intermediates formed upon condensation of acetaldehyde with the amino group in PE.Proton diffusion along water molecules above the polar heads of phospholipids spread in monolayers has been experimentally found to be 20times faster than in the bulk water phase,which has been ascribed to the presence of the hydrogen bond network along polar heads capable of supporting a rapid “hop and turn”mechanism of proton conduction along the interface.70,71As can be seen in Table 1,the free energy barrier for dehydration of the carbinolamine was 18.88kcal/mol;therefore,this reaction is the rate-determining step of the process.Similar conclusions for this type of reaction have been drawn from the-oretical and experimental results for other,simpler systems.16,17,72The iminium ion product (E9)involved in the first part of this step has a high free energy relative to the other intermediates of the product of the Schiff base formation;this testifies to the increased chemical reactivity of the nitrogen atom (N3)by effectSCHEME 1:ContinuedaDotted lines represent hydrogen bonds.TABLE 1:Relative Energies and Free Energies (at 1atm and 298K)of the Structures of the Reaction Pathstructure ∆E (kcal/mol)∆G (kcal/mol)E10.0000.000TS2-0.927 2.508E3-8.420-8.489TS4-3.262-5.172E5-8.825-7.000TS6 1.475-3.001E7-8.001-5.801TS814.92313.080E97.701 6.981TS1015.96511.625E11-3.709-5.744Figure 2.Free energy (1atm,298K)profile for the reaction.Reactivity of a Phospholipid Monolayer Model J.Phys.Chem.B,Vol.114,No.48,201015883of major geometric changes during rehybridization to the iminium ion.In addition,the nitrogen carries positive charge and may therefore interact electrostatically with the phosphate anion group in DAPE.Iminium ions are known to be unstable except in heterocyclic systems,73and on the basis of the cal-culated free energy profile (Figure 2),conversion into the neutral Schiff base in the following step is a favorable process.The free energy barrier for the dehydration step in our system is slightly high relative to other previously studied systems.This may be a result of other chemical groups providing additional assistance in these reactions.Theoretical studies on the pyridoxal 5′-phosphate (PLP)-dependent transamination of R -amino acids,where the relative energy barrier in the gas phase is 16.9kcal/mol,have revealed that a carboxylic group in the amino acid acts as a proton donor facilitating water elimination and also that a phenol group in PLP helps stabilize the system.69A DFT study of the Schiff base formation between a pyridoxamine analogue and acetaldehyde or glycolaldehyde in the gas phase provided relative energy barriers from 10to 15kcal/mol;a phenolic hydroxyl group was found to act as a proton donor to the carbinolamine hydroxyl group to produce the leaving water molecule.17Phospholipids also possess a phosphate group that may play a role in this reaction.We probed a phosphate group as proton acceptor;unfortunately,we obtained no stable species because of its too low p K a ,the experimental value for which in PE is 0.5.74The phosphate anion might enhance Schiff base formation via a neighboring catalyst effect;in fact,we found it to form hydrogen bonds with water molecules in the network connecting donor and acceptor protons,and amino groups of DAPE (Figure 1),in different steps of the studied mechanism.Phosphate groups can play three different roles in this context,namely,(a)facilitating accumulation of H 2O on the membrane surface and raising local concentrations as a result (as found in previous studies,negatively charged phosphate groups are tightly solvated by an average of four water molecules each),75(b)polarizing water bonds through interaction with them (P-bound water molecules tend to orient their dipoles with their positive ends pointing at negatively charged phosphate groups,thereby resulting in net orientational “polarization”,76which may facilitate the role of solvation water molecules as bridges for the proton exchange between donor and acceptor protons in the reaction),and (c)exerting a passive catalytic effect by stabilizing charge in various reaction intermediates through direct electro-static interactions with the positively charged groups produced in the different reaction steps.The Schiff base formation reaction between a model surface cell membrane and an acetaldehyde provides a simple means for illustrating the potential of phospholipid groups in cell membranes and their solvating water molecules to enhance a reaction via a neighboring catalyst effect.Therefore,it may help to shed some light on cell membrane glycation.The use ofPBCsFigure 3.Pathway for protonation of zwitterionic form of the carbinolamine molecule.(a)Zwitterionic molecule (E3),(b)TS (TS4),and (c)positive charged product(E5).Figure 4.Pathway for dehydration of carbinolamine molecule.(a)Neutral carbinolamine molecule (E7),(b)TS (TS8),and (c)iminium ion product (E9).15884J.Phys.Chem.B,Vol.114,No.48,2010Solı´s-Calero et al.in our computations proved essential as it allowed us not only to model a phospholipid surface but also to study its reactivity at the DFT level.This methodology may also facilitate computational studies on Schiff base formation between sugars and potential inhibitors at membranes. Acknowledgment.This work was funded by the Spanish Government in the framework of Project CTQ2008-02207/BQU. We gratefully acknowledge technical support and extensive discussions with Dr.A.Herna´ndez-Laguna(Estacio´n Experi-mental del Zaidı´n,CSIC,Granada).C.S.-C.acknowledges a MAE-AECI fellowship from the Spanish Ministry of Foreign Affairs and Cooperation.We are grateful to Centro de Ca´lculo de Computacio´n de Galicia(CESGA),and the Centro de Ca´lculo de Computacio´n de Catalun˜a(CESCA),for access to their computational facilities.Supporting Information Available:Geometrical parameters of structures presented in Scheme1and videos of some proton transferences.This material is available free of charge via the Internet at .References and Notes(1)Luthra,M.;Balasubramanian,D.J.Biol.Chem.1993,268,18119–18127.(2)Li,Y.;Cohenford,M.A.;Dutta,U.;Dain,J.A.Anal.Bioanal. Chem.2008,390,679–688.(3)Bucala,R.;Makita,Z.;Koschinsky,T.;Cerami,A.;Vlassara,H. 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