N-linked glycan structures of a mouse monoclonal antibody produced from tobacco BY2 suspension-cultured cells

cDNA encoding H- and L-chains from a mouse monoclonal antibody was introduced into tobacco BY2 cells, and the resulting sugar chain structures of plant-produced antibodies were analyzed by a combination of HPLC, exoglucosidase digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were Man(5-6)GlcNAc2 (22.3%), GlcNAcMan5GlcNAc2 (3.1%), GlcNAcMan3FucXylGlcNAc2 (24.4%), GlcNAcMan3XylGlcNAc2 (17.8%), Man3FucXylGlcNAc2 (24.3%), and Man3XylGlcNAc2 (8.1%). The major glycan structures of the antibodies produced by transgenic suspension-cultured cells contain typical plant bisecting beta(1,2)-xylose and alpha(1,3)-fucose residues, suggesting the posttranslational modification of a recombinant antibody in the late Golgi apparatus.     

Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides

N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP-N-acetylglucosamine to the alpha1,3-linked mannose on Man5GlcNAc2 (M5), is a critical enzyme for the synthesis of high-mannose-type to complex-type glycan structures in N-linked glycan processing. We developed a large-scale preparation system for recombinant human GnT-I (hGnT-I) using the maltose binding protein (MBP) fusion system to facilitate the chemoenzymatic route for complex-type N-linked glycan synthesis. MBP-fused GnT-I was purified by affinity chromatography on an amylose resin column. The relative activity of MBP-fused GnT-I toward high-mannose-type N-linked oligosaccharides was 100% for Man5GlcNAc2, 52% for Man3GlcNAc2, 17% for Man6GlcNAc2. MBP-fused GnT-I exhibited optimal activity at pH 6.5-9.5 and was more active between pH 6.5-9.0. The optimum temperature for MBP-fused GnT-I activity was 40 degrees C, but the enzyme was active between 0-70 degrees C. Mn2+ and Co2+ were critical for the enzyme activity, while Zn2+ and Ca2+ inhibited the activity. Kinetic analysis of the purified enzyme showed an apparent K(m) value of 0.483 mM and a V(max) of 101 nmol/mg/min for M5. Immobilization of MBP-fused GnT-I on the amylose resin led to an 80% yield of the high mannose-type-of oligosaccharide.     

Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system

Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P₁₀ and Polyhedrin promoters in the pFastBac™ dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAb^I) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAb^I from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAb^I had insect specific glycan structures that differed from their mammalian counterparts, mAb^I similarly interacted with CD64 (FcγRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.     

Dietary N‐Glycans from Bovine Lactoferrin and TLR Modulation

1 Scope

Bovine lactoferrin (bLF) is an ingredient of food supplements and infant formulas given its antimicrobial and antiviral properties. We modified bLF enzymatically to alter its N‐glycosylation and to isolate the glycan chains. The aims of this study include (1) to evaluate whether such derivates induce responses via pattern recognition receptors namely Toll‐like receptors (TLRs) and (2) to relate those responses to their different glycosylation profiles.

2 Methods and results

The unmodified and modified bLF fractions are incubated with reporter cell lines expressing pattern recognition receptors. Afterwards, we screen for TLRs and analyze for nuclear factor kappa—light‐chain enhancer of activated B cells (NF‐κB) activation. Activation of reporter cell lines show that signaling is highly dependent on TLRs. The activation pattern of bLF is reduced with the desialylated form and increased with the demannosylated form. In reporter cells for TLR, bLF activate TLR‐4 and inhibit TLR‐3. The isolated glycans from bLF inhibit TLR‐8. TLR‐2, TLR‐5, TLR‐7, and TLR‐9 are not significantly altered.

3 Conclusion

The profile of glycosylation is key for the biological activity of bLF. By understanding how this affects the human defense responses, the bLF glycan profile can be modified to enhance its immunomodulatory effects when used as a dietary ingredient.     

N-糖基化对β-甘露聚糖酶在毕赤酵母中异源表达的影响

为研究N-糖基化对里氏木霉β-甘露聚糖酶(β-mannanase,Man1)在毕赤酵母GS115中异源表达的影响,采用定点突变的方法,将Man1上3个N-糖基化修饰位点(N131、N158和N329)上的天冬酰胺用中性谷氨酰胺取代。结果发现,N-糖基化位点的突变对Man1的转录水平无明显影响,突变后获得的Man1表观分子质量有轻微下降,与Man1相比,3个突变体N131、N158和N329的甘露聚糖酶活力分别降低了85.43%和79.48%和16.3%;而热稳定性分别提高了7.87%、13.5%和15.37%。由此可见,N-糖基化修饰对于β-甘露聚糖酶的高效表达是必需的,其中N131和N158糖基化位点尤为重要,但是会稍微降低β-甘露聚糖酶的热稳定性。     

糖基化方法对于大豆7S球蛋白糖基化反应及产物的影响

针对传统的两种糖基化接枝方法和改进方法从糖基化反应程度进行系统比较,并对产物氨基酸组成进行分析,以期进一步揭示蛋白质糖基化方法对于糖基化反应过程及产物的影响机制。本文利用干热法、湿热法和加压辅助湿热三种糖基化方法,对大豆7S球蛋白和葡聚糖的糖基化产物从反应程度、氨基酸组成等方面进行研究发现,结果表明:三种制备方法的反应程度高低顺序依次为:湿热法>加压辅助湿热法>干热法,说明压力能够对Maillard反应的进行具有一定的抑制作用,可以控制反应向理想阶段进行。大豆7S球蛋白和葡聚糖的糖基化主要发生在蛋白质肽链上的赖氨酸和精氨酸侧链上的自由氨基,干热法与其它两种方法相比糖链更有易于和精氨酸侧链上的自由氨基发生共价交联,而湿热法和加压辅助湿热法相比糖链更易于和赖氨酸侧链上的自由氨基发生共价交联。     

Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris

We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris. rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial. The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT). In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns. The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated. The neutral oligosaccharides were thought to be Man(9-12)GlcNAc(2) as their major components. The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column. Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components. The mannose residues at the non-reducing end(s) of Man(9-12)GlcNAc(2) were phosphomannosylated or phosphorylated and these are the major components. Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels.     

Characterization of glycans in a lactoferrin isoform, lactoferrin-a

The presence of glycan at Asn-281 in bovine lactoferrin-a, which has a higher molecular weight than regular lactoferrin-b, was found in our previous study. The present work was performed to clarify the structures of the glycans linked to the five N-glycosylation sites in lactoferrin-a and to compare them with those of glycans linked to lactoferrin-b. In lactoferrin-a, the glycans linked to Asn-233 and Asn-545 were of the high-mannose type, whereas those present at Asn-368 and Asn-476 were complex-type ones. These glycans possessed heterogeneous structures. A comparative study of the glycans on bovine lactoferrin-a and bovine lactoferrin-b by HPLC showed that the structures of the glycans linked to Asn-368, Asn-476, and Asn-545 were very similar, the exception being the glycan linked to Asn-233. In addition, analysis of the structure of the glycan bound to Asn-281 present only in lactoferrin-a showed it possessed the heterogeneous structure of a complex-type glycan in which the structures Man3GlcNAc2, Man3GlcNAc4, Man3GlcNAc4Fuc are suggested to be present based on HPLC retention times only.     

Phosphorylation and glycosylation isoforms of bovine κ-casein variant E in homozygous Swedish Red cow milk detected by liquid chromatography-electrospray ionization mass spectrometry

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ～70%; where P indicates phosphorylation from single to triple phosphorylation (1–3P), and G indicates glycosylation from single to triple glycosylation (1–3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ～12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ～0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences.     

N-糖酰胺酶PNGase H+最小糖结构作用底物的研究

为探究N-糖酰胺酶的最小糖结构作用底物,以丹磺酰氯标记的复杂型七糖糖肽标准品为初始底物,运用酶法合成的方法来获得目标底物,分别使用β-N-乙酰氨基己糖苷酶、α-甘露糖苷酶、β-甘露糖苷酶和β-半乳糖基转移酶,制备出五种不同糖结构的糖肽底物,将制备的目标底物应用于N-糖酰胺酶PNGase H+最小糖结构作用底物的研究中。通过高效液相色谱检测PNGase H+对这5种糖肽底物的酶解效果。结果表明,得到五种不同结构的糖肽底物,Dabsyl-Gly-Glu-Asn-(GlcNAc2Man3)-Arg、Dabsyl-Gly-Glu-Asn-(GlcNAc2Man)-Arg、Dabsyl-Gly-Glu-Asn-(GlcNAc2)-Arg、Dabsyl-Gly-Glu-Asn-(GlcNAc)-Arg及Dabsyl-Gly-Glu-Asn-(GlcNAc2Gal)-Arg,浓度分别为8.3、7.0、6.5、4.5、6.5 pmol/μL。N-糖酰胺酶PNGase H+能够作用糖单元数为二、三和五的糖肽底物,而对含有单个N-乙酰葡糖胺的糖肽几乎没有作用,进而推断PNGase H+的最小糖结构作用底物为带有N-乙酰壳二糖的糖肽。     

The pH-dependent thermal and storage stability of glycosylated caseinomacropeptide

Bioactive properties of bovine glycosylated caseinomacropeptide (gCMP) like antibacterial effects are reported to be closely associated with their structure in terms of attached glycans. Technological properties are also influenced by the carbohydrate side chains. However, during product manufacturing gCMP can be modified due to processing. Processing conditions, which influence the degree of glycosylation of gCMP lead to alterations of bioactivity and techno-functional properties of gCMP and accordingly gCMP-containing products. Hence, gCMP was studied for its glycan stability during heat treatment and storage under different pH values. Process stability (preservation of native protein structure in terms of attached glycans) was analysed by quantifying the release of the terminal carbohydrate, N-acetylneuraminic acid (Neu5Ac), from gCMP. The results clearly showed that the thermal stability of gCMP is strongly influenced by pH. When the pH was decreased from 7 to 2, reduced stability was found even at low heating temperatures. Minimal destabilisation effects were found at neutral pH. Similar observations were found during storage of gCMP. Neu5Ac was released after six days of storage, with a maximum release of 30% at pH 2. Acidic pH conditions were responsible for the hydrolysis of the glycans from the peptide backbone during heat treatment and storage.     

大豆蛋白水解物中肽分子分布的研究

研究采用超滤法与凝胶过滤色谱（GFC）相结合实验方法就大豆蛋白酶解产物寡肽混合物的分子量的分布状况进行了测定,研究结果表明,木瓜蛋白酶与Asl.398蛋白酶的酶解物中以分子量在1000D以下的小肽为主：木瓜蛋白酶解产物中分子量1000D以下的占63.9%,1000-2000D之间的4.64%,2000-4000D之间的占8.21%,4000-10000D之间的占8.20%,分子量10000D以上的占15%;Asl.398蛋白酶酶解物中分子量1000D以下的占72.1%,1000D-2000D的占6.42%,2000D-4000D之间的占2.5%,4000-10000D之间的占3.92%,10000D以上的占15%。     

糖基化反应对蛋清蛋白质凝胶性的影响

为研究糖基化反应提高蛋清蛋白质凝胶性的机理,利用氨基酸分析仪、苯酚硫酸法、奥氏黏度计、静态激光散射仪对糖基化蛋清蛋白质氨基酸组成、总糖含量、特性黏度、粒径分布进行测定与分析。结果显示：糖基化反应可使蛋清蛋白质的凝胶强度和持水性分别提高91.7%和35.2%,且二者均在反应4d后达到最高值;糖基化处理5d后,蛋清蛋白质的赖氨酸含量相对降低28.42%,总糖含量增至2.18%;特性黏度随反应时间的延长增幅较小;糖基化蛋白粒径分布在0.1～50.0μm之间,糖基化2d内,粒径分布由小粒径峰向大粒径峰转变;糖基化2d后,粒径分布变化不明显。     

Large-scale preparation and glycan characterization of sialylglycopeptide from bovine milk glycomacropeptide and its bifidogenic properties

Bovine glycomacropeptide (GMP) is a 7,000-Da glycopolypeptide released from κ-casein during cheese making. The O-glycan chains linked to GMP have many biological activities, but their utilization for nutraceutical products is limited due to their low content. To concentrate the functional glycan chains of GMP, we prepared sialylglycopeptide concentrate (SGC) from GMP-containing whey protein concentrate via proteolytic digestion of peptide chains and concentration of sialylglycopeptide by ultrafiltration using membranes with a molecular weight cut-off of 1,000 Da. The abundant saccharides detected in the prepared SGC were N-acetylneuraminic acid (Neu5Ac: 32.3% wt/wt), N-acetylgalactosamine (11.3%), and galactose (10.2%), which constitute O-glycans attached to GMP. The Neu5Ac content in SGC was found concentrated at approximately 4.8-fold of its content in GMP-containing whey protein concentrate (6.8%). Structural analysis of O-glycopeptides by liquid chromatography tandem mass spectrometry identified 88 O-glycopeptides. Moreover, O-acetylated or O-diacetylated Neu5Ac was detected in addition to the previously characterized O-glycans of GMP. Quantitative analysis of O-glycan in SGC by fluorescence labeling of chemically released O-glycan revealed that a disialylated tetrasaccharide was the most abundant glycan (76.6% of the total O-glycan). We further examined bifidogenic properties of SGC in vitro, which revealed that SGC served as a more potent carbon source than GMP and contributes to the growth-promoting effects on certain species of bifidobacteria. Overall, our study findings indicate that SGC contains abundant O-glycans and has a bifidogenic activity. Moreover, the protocol for the preparation of SGC described herein is relatively simple, providing a high yield of glycan, and can be used for large-scale preparation.     

Variations and distributions of O-glycosidically linked sugar chains in bovine kappa-casein.

The variation and distribution of O-glycosidically linked sugar chains in kappa-casein from bovine mature milk were analyzed by HPLC, fast atom bombardment mass spectrometry, and 13C nuclear magnetic resonance. A monosaccharide alditol (N-acetylgalactosaminitol) and four oligosaccharide alditols [a neutral disaccharide (galactosyl-beta 1-3N-acetylgalactosaminitol), two acidic trisaccharides (straight chain: N-acetylneuraminyl alpha 2-3galactosyl-beta 1-3N-acetylgalactosaminitol and branch chain: galactosyl beta 1-3(N-acetylneuraminyl alpha 2-6)N-acetylgalactosaminitol), and an acidic tetrasaccharide(N-acetylneuraminyl alpha 2-3galactosyl-beta 1-3(N-acetylgalactosaminyl alpha 2-6)N-acetylgalactosaminitol] were identified as sugar chains linking on normal bovine kappa-casein. The most dominant sugar chain was an acidic tetrasaccharide. The four oligosaccharide chains were identical to those already found, but N-acetylgalactosamine was a newly identified sugar chain linking on kappa-casein. The distribution of monosaccharide, disaccharide, trisaccharide (straight), trisaccharide (branched), and tetrasaccharide chains were determined by HPLC to be .8, 6.3, 18.4, 18.5, and 56.0%, respectively (means of five kappa-caseins).     

Short communication: Application of proteomics for characterization of caseinomacropeptide isoforms before and after desialidation

Caseinomacropeptide (CMP) is an important polypeptide found in cheese whey that has various biological activities and functional properties. Because sialylations play an important role in the functional properties of CMP, the aim of the present work was to characterize CMP isoform heterogeneity in a commercial glycosylated CMP (gCMP) isolate using liquid chromatography– and gel-based proteomics before and after desialidation. Using 2-dimensional gel electrophoresis (2-DGE), we observed a shift in isoelectric point (pI) after enzymatic desialidation, indicating that sialylated gCMP were located at pI 3.0 to 3.1, but desialylated gCMP had repositioned to pI 3.7 to 3.9. We used liquid chromatography/mass spectroscopy (LC-ESI/MS) for further analysis of the glycan chains of gCMP. In total, we identified 19 CMP isoforms, of which 13 were glycosylated and 6 were nonglycosylated. We also detected 3 genetic variants (A, B, and E), with up to 3 glycosylations attached per gCMP. Further, we identified up to 4 isomers, reflecting different sites of glycosylation in the peptide backbone of these isoforms. The dominating gCMP isoform of genetic variant E appeared to contain 4 N-acetyl-neuraminic acid (NeuAc) residues, whereas the dominating gCMP isoforms of variants A and B appeared to contain 2 NeuAc. The identification revealed conversions of isoforms containing different combinations of genetic variants and degrees of glycosylation, sialylation, and phosphorylation to various desialylated counter-isoforms. The content of sialylated gCMP relative to the total CMP content was 37% (wt/wt), which decreased to 1.5% after sialidase treatment, indicating 96% effectivity of the desialidation. This approach can be valuable for future functionality studies specifically addressing the importance of NeuAc.     

抗莱克多巴胺单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定

莱克多巴胺(RAC)经化学修饰引入功能基团羧基,合成半抗原RAC- 戊二酸酐半醛化合物(RAC-SA),用混合酸酐法(MA)将RAC-SA 偶联于牛血清白蛋白(BSA),合成人工抗原BSA-RAC,用红外(IR)、紫外(UV)和凝胶电泳(SDS-PAGE)对其进行鉴定;用BSA-RAC免疫Balb/C 小鼠,细胞融合技术建立抗RAC的单克隆抗体(RAC mAb)杂交瘤细胞株,体内诱生腹水法制备RAC mAb,并鉴定其免疫学特性。结果表明,BSA-RAC 偶联成功,偶联率为24.5:1;筛选出3F10、3H12、4D8 共3 株杂交瘤细胞,其中最好的4D8 株间接ELISA 效价细胞培养上清为1:1.28 × 103,腹水为1:6.4 × 105,同种型为IgG1/κ,亲和常数(Ka)为1.65 × 1010L/mol,对RAC 的半数抑制浓度(IC50)为5.7ng/ml,与多巴酚丁胺的交叉反应率(CR)为22.3%,与其他化合物无CR。     

糖基化提高大豆分离蛋白凝胶性的工艺条件

以提高大豆分离蛋白的凝胶强度为目的,采用添加D(+)木糖和黄原胶进行糖基化改性处理,中心组合设计模型对大豆分离蛋白共价改性工艺条件进行优化,测定并分析了改性复合物在各个条件下的凝胶强度。结果表明:适宜反应条件为反应温度87.27℃、反应时间40 min、复合糖添加量3.9%、糖胶比2.81∶1,此条件下凝胶强度可达到91.35 g,较未改性大豆分离蛋白提高78%。     

食品中蛋白质糖基化接枝的研究进展

在食品领域,基于Maillard反应机理的蛋白质和多糖糖基化方法是蛋白质改性方法的一种绿色有效方法,属于蛋白质化学改性范畴,在此反应中,蛋白质与糖发生共价接枝,不需要添加任何化学试剂作为催化剂,仅加热就可使反应自发进行,以往的研究表明,蛋白质的糖基化是提高蛋白质功能特性及其他应用性质的有效途径之一。文章系统介绍了蛋白质糖基化的结构、反应机理、糖链在蛋白质糖基化中的主要作用、糖基化方法,并对蛋白质糖基化的研究方向进行了探讨和展望。一方面,由于蛋白质糖基化方法的一些局限性,难以实现可控性和工业性、规模化应用,如何更好地利用跨学科的思路,对现有糖基化方法进行研究探讨,在达到糖基化过程的可控性和工业化方面将是关注热点。另一方面,如何更好地拓展糖基化产物的应用性,也是今后的研究热点。     

Hemicellulosic contamination of acid detergent residues and their replacement by cellulose residues in cell wall analysis

The carbohydrate compositions of acid detergent residues, prepared from 26 plant samples, ranging from fruits, vegetables, legumes, forages and woods, were determined and up to 15.4% of the sugar units present were neutral sugars other than D-glucose and derived from hemicelluloses. Similar analyses on cellulose residues, prepared by the acetic acid-nitric acid method, showed up to 9.6% of these residues were derived from hemicellulosic contaminants. These results suggest that care should be taken before using acid detergent residue values to calculate the cellulose and hemicellulose content of plant materials. It is observed that more accurate results will be obtained by using values for cellulose residues and this method is recommended even though it is not absolutely accurate, particularly when analysing plant materials with a high cell wall content.