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Hydroxyproline Assay Using NaBr/NaOCl
使用 NaBr/NaOCl进行羟脯氨酸检测   

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Abstract

Hydroxyproline (Hyp) is a major constituent of a relatively few proteins that are major structural components of the extracellular matrix and primary cell wall of animals and plants respectively. Significant amounts of the cyclic amino acids proline and hydroxyproline decrease polypeptide flexibility; thus proline/hydroxyproline-rich proteins are ideal scaffold components. Collagens typify animal tissues but extensins, arabinogalactan proteins (AGPs) and their close relatives, collectively referred to as hydroxyproline-rich glycoproteins (HRGPs), typify plants (Lamport et al., 2011). While collagens are minimally glycosylated generally via a galactosyl hydroxylysine linkage, plant HRGP glycosylation involves short neutral oligosaccharides (in extensins) or much larger acidic polysaccharide substituents (in AGPs) O-linked via the hydroxyproline hydroxyl group. Hydroxyproline assay is thus an integral part of their characterization and dominates the biochemical properties of these glycoproteins. The colourimetric assay described here quantifies free hydroxyproline (e.g. released by acid hydrolysis) based on Kivirikko and Liesmma (1959) with hypobromite as an oxidant but modified by avoiding the use of hazardous liquid bromine. A number of oxidants have been used over the years, Vogel (1961, page 395) explains the preference for hypobromite as follows: “Hypochlorites tend to react slowly with reducing agents. Hypobromites although rather unstable when prepared directly from bromine and alkali, often react more rapidly; it is therefore advantageous to produce hypobromite in situ by adding an excess of bromide to the sample of hypochlorite:” OCl- + Br- → OBr- + Cl- “By this means the relative stability of hypochlorite is combined with the more effective oxidizing properties of hypobromite.

Materials and Reagents

  1. NaOCl (Lab bleach)
  2. NaOH
  3. NaBr
  4. 6 N HCl
  5. Dilute hypobromite
  6. p.dimethylaminobenzaldehyde (Sigma-Aldrich, catalog number: 156477 )
  7. n.propanol (Sigma-Aldrich, catalog number: 402893 )

Equipment

  1. 2 ml screw-cap microtube (SARSTEDT AG)
  2. Microplate reader or spectrophotometer

Procedure

  1. Prepare dilute sodium hypobromite (NaOBr) from NaOCl, mix equal volumes (a) + (b) (e.g. 5 ml each) (prepare fresh weekly-store at 4 °C).
    1. Add 775 μl lab bleach to 10 ml 4% NaOH (fresh weekly).
    2. Prepare 100 mM NaBr (1.03 g in 100 ml 4% NaOH) (stable).
  2. Add 250 μl aqueous sample to 2 ml screw-cap microtubes (e.g. SARSTEDT AG).
  3. Add 500 μl dilute hypobromite to:
    1. Analysis samples each in 250 μl distilled water.
    2. Hyp standards of 2.5, 5.0, 7.5 and 10 μg, each in 250 μl distilled water.
    3. A reagent blank contains reagents plus an additional 250 μl distilled water.
  4. Mix and leave for 5 min to oxidize at room temperature.
  5. Add 250 μl 6 N HCl.
  6. Add 500 μl 5% p.dimethylaminobenzaldehyde in n.propanol (total volume = 1.5 ml).
  7. Mix and heat at 70 °C for 15 min, then cool in ice-water.
  8. Measure absorbancy of samples and standards at 560 nm against the reagent blank. e.g. 10 μg Hyp → ~680 mAUs
  9. Construct the standard curve and calculate sample values by interpolation.

Recipes

  1. This assay determines free hydroxyproline, best prepared by hydrolysis peptide bonds in the sample to be analysed (6 N HCl at 110 °C for 18 h) followed by removal of HCl in vacuo and redissolving the hydrolysate in distilled water. Interestingly free O-glycosylated Hyp can be assayed directly.
  2. Use aqueous Hyp standards over a 2.5 to 10 μg range. It is convenient to prepare a Hyp standard of 100 μg/ml in distilled water stored frozen; mix well after thawing! The reagent blank contains all the reagents with distilled water as a substitute for the sample.
  3. 5% w/w p.dimethylaminobenzaldehyde in n-propanol.

Acknowledgments

This protocol is adapted from Kivirikko and Liesmaa (1959) and Lamport et al. (2012).

References

  1. Kivirikko, K. I. and Liesmaa, M. A. (1959). A colorimetric method for determination of hydroxyproline in tissue hydrolysates. Scandinavian J Clin Lab 11(2): 128-133.
  2. Lamport, D. T., Kieliszewski, M. J., Chen, Y. and Cannon, M. C. (2011). Role of the extensin superfamily in primary cell wall architecture. Plant Physiol 156(1): 11-19.
  3. Vogel, A. I. (1961). A text-book of quantitative inorganic analysis. 1-1216, Third Edition. Publisher: Longmans.

简介

羟脯氨酸(Hyp)是相对较少的蛋白质的主要成分,其分别是动物和植物的细胞外基质和原代细胞壁的主要结构组分。显着量的环状氨基酸脯氨酸和羟脯氨酸降低多肽的柔性;因此富含脯氨酸/羟脯氨酸的蛋白质是理想的支架组分。胶原代表动物组织,但是延伸蛋白,阿拉伯半乳聚糖蛋白(AGP)及其近亲,统称为富含羟脯氨酸的糖蛋白(HRGP),代表植物(Lamport等人,2011)。虽然胶原通常通过半乳糖基羟基赖氨酸键最小程度地糖基化,但植物HRGP糖基化包括通过羟基脯氨酸羟基O-连接的短中性寡糖(在延伸素中)或更大的酸性多糖取代基(在AGP中)。羟脯氨酸测定因此是它们的表征的组成部分并且主导这些糖蛋白的生物化学性质。这里描述的比色测定基于Kivirikko和Liesmma(1959)用次溴酸盐作为氧化剂定量 羟脯氨酸(例如通过酸水解释放的)通过避免使用危险的液体溴改性。多年来已经使用了许多氧化剂,Vogel(1961,第395页)解释了对次溴酸盐的偏好如下:"次氯酸盐倾向于与还原剂缓慢反应。次溴酸盐虽然在直接由溴和碱制备时相当不稳定,但通常反应更快;因此有利的是通过向次氯酸盐样品中加入过量的溴化物原位生产次溴酸盐:"OCl + Br →OBr > + Cl - "这意味着次氯酸盐的相对稳定性与次溴酸盐的更有效的氧化性质相结合。"

材料和试剂

  1. NaOCl(实验室漂白)
  2. NaOH
  3. NaBr
  4. 6 N HCl
  5. 稀释次溴酸盐
  6. 二甲基氨基苯甲醛(Sigma-Aldrich,目录号:156477)
  7. 正丙醇(Sigma-Aldrich,目录号:402893)

设备

  1. 2ml螺旋盖微量管(SARSTEDT AG)
  2. 酶标仪或分光光度计

程序

  1. 从NaOCl制备稀的次溴酸钠(NaOBr),混合等体积(a)+(b)(例如每次5ml)(在4℃下制备新鲜的每周储存)。
    1. 加入775μl实验室漂白剂到10 ml 4%NaOH(每周新鲜)
    2. 制备100mM NaBr(1.03g在100ml 4%NaOH中)(稳定)
  2. 将250μl水样加入2ml螺旋盖微量管(例如SARSTEDT AG)中。
  3. 加入500μl稀二次沸石至:
    1. 分析样品在250μl蒸馏水中
    2. Hyp标准为2.5,5.0,7.5和10μg,每个在250μl蒸馏水中
    3. 试剂空白包含试剂加另外250μl蒸馏水
  4. 混合并离开5分钟,在室温下氧化
  5. 加入250μl6N HCl。
  6. 加入500μl5%对二甲基氨基苯甲醛的正丙醇(总体积= 1.5ml)
  7. 混合并在70℃加热15分钟,然后在冰水中冷却
  8. 测量样品和标准物在560nm下对试剂空白的吸光度。例如10μgHyp→〜680mAU。
  9. 构建标准曲线并通过插值计算样本值。

食谱

  1. 该测定法确定最好通过待分析样品中的水解肽键(6N HCl,在110℃下18小时)制备的游离羟脯氨酸,随后除去HCl < 并在蒸馏水中再溶解水解产物。 有趣的是,可以直接测定游离 O-糖基化的Hyp。
  2. 使用2.5至10μg范围内的含水Hyp标准品。 在冷冻储存的蒸馏水中制备100μg/ml的Hyp标准是方便的; 混合后解冻! 试剂空白包含所有用蒸馏水代替样品的试剂
  3. 5%w/w对二甲基氨基苯甲醛在正丙醇中

致谢

该协议改编自Kivirikko和Liesmaa(1959)和Lamport等人(2012)。

参考文献

  1. Kivirikko,K.I.和Liesmaa,M.A。(1959)。 用于测定组织水解产物中羟基脯氨酸的比色法。Scandinavian J Clin Lab 11(2):128-133
  2. Lamport,D.T.,Kieliszewski,M.J.,Chen,Y.and Cannon,M.C。(2011)。 延伸蛋白超家族在原代细胞壁结构中的作用植物 Physiol 156(1):11-19。
  3. Vogel,A.I。(1961)。 定量无机分析的教科书。 1-1216,第三版。 出版商:Longmans。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lamport, D. T. (2013). Hydroxyproline Assay Using NaBr/NaOCl. Bio-protocol 3(19): e917. DOI: 10.21769/BioProtoc.917.
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