基于2,4-二硝基氟苯标记氨基酸的高效液相色谱法测定脂肽含量
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摘要
本文将提取的一种脂肽(表面活性素)水解成其组成的氨基酸,以2,4-二硝基氟苯标记产生的氨基酸后,用高效液相色谱(HPLC)分离标记的目标氨基酸(亮氨酸),记录色谱图,分析氨基酸的硝基苯标记物的积分面积与氨基酸的关系。结果表明,脂肽中的氨基酸与标记产物的积分面积有很好的线性关系(R~2>0.99),据此可以测定脂肽的含量。测定了样品中表面活性素的含量,其相对标准偏差为6.5%,样品回收率92.6%(n=5)。因大部分脂肽均含有一个或多个亮氨酸或异亮氨酸,所含氨基酸的种类和数量是一定的,故该方法还可以用于测定其它类别脂肽的含量。
Determination of lipopeptides is important for their application since lipopeptides are important biosurfactants which may be used in many commercial fields such as medicine,food and cosmetics industries.In this work,a lipopeptide(extracted surfactin) was firstly hydrolyzed to amino acids,and the produced amino acids were then labeled by 2,4-dinitrofluorobenzene(FDNB) and finally analyzed by high performance liquid chromatography(HPLC).The relationship between integration areas of labeled products and amino acid amounts was established from the chromatograms.Results showed that there was good linear relationship between the amount of amino acids in lipopeptides and the integration areas of labeled amino acids(n=7,linear regression coefficient was more than 0.99).The relative standard deviation of 6.5% was observed with a recovery of 92.6%(n=5) when the method was used to determine surfactin sample.The method can also be applied to the determination of other lipopeptides quantitatively since most lipopeptides contain one or more leucine or isoleucine which are used to be labeled.
Determination of lipopeptides is important for their application since lipopeptides are important biosurfactants which may be used in many commercial fields such as medicine,food and cosmetics industries.In this work,a lipopeptide(extracted surfactin) was firstly hydrolyzed to amino acids,and the produced amino acids were then labeled by 2,4-dinitrofluorobenzene(FDNB) and finally analyzed by high performance liquid chromatography(HPLC).The relationship between integration areas of labeled products and amino acid amounts was established from the chromatograms.Results showed that there was good linear relationship between the amount of amino acids in lipopeptides and the integration areas of labeled amino acids(n=7,linear regression coefficient was more than 0.99).The relative standard deviation of 6.5% was observed with a recovery of 92.6%(n=5) when the method was used to determine surfactin sample.The method can also be applied to the determination of other lipopeptides quantitatively since most lipopeptides contain one or more leucine or isoleucine which are used to be labeled.
引文
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[2] LIU X Y,YANG S Z,MU B Z.Biotechnology Bulletin(刘向阳,杨世忠,牟伯中.生物技术通报),2005,(4):18.
[3] Yang S Z,Liu J F,Mu B Z.12th International Congress on Amino Acids,Peptides and Proteins.Beijing:1-5 Aug,2011.Amino Acids,2011,41(Suppl 1):S40.
[4] Yu G Y,Sinclair J B,Hartman G L,Bertagnolli B L.Soil Biol Biochem,2002,34:955.
[5] Morikawa M,Hirata Y,Imanaka T.Biochim Biophys Acta,2000,1488:211.
[6] Kikuchi T,Hasumi K.Biochim Biophys Acta,2002,1596:234.
[7] Olivera N L,Commendatore M G,Moran A C,Esteves J L.J Ind Microbiol Biotech,2000,25:70.
[8] Mulligan C N,Yang R N,Gibbs B F.J Hazard Mater,2001,85:111.
[9] Lin S C,Chen Y C,Lin Y M.J Chromatogr A,1998,825:149.
[10] Yang S Z,Chen T,Mu B Z,et al.Chinese Chemical Letters,2006,17(4):517.
[11] CHEN T,YANG S Z,MU B Z.Oilfield Chemistry(陈涛,杨世忠,牟伯中.油田化学),2004,21(4):385.
[12] Zhao Y,Yang S Z,Mu B Z.Analytical Science,2012,28(8):789.
[13] ZHAO Y,YANG S Z,MU B Z.Chemistry Bulletin(赵越,杨世忠,牟伯中.化学通报),2013,76(2):135.
[14] Arima K,Kakinuma A,Tamura G.Biochem Biophys Res Commun,1968,31:488.
[15] Satake K,Matsuo A,Take T.The Journal of Biochemistry,1965,58(1):90.
[16] Liu J F,Yang J,Yang S Z,et al.Applied Biochemistry and Biotechnology,2012,166(3):2091.
[17] Liu X Y,Haddad N I A,Yang S Z,Mu B Z.Protein & Peptide Letters,2007,14(8):766.
[18] Li Y M,Yang S Z,Mu B Z.Analytical Letters,2010,43(6):929.
[19] Wang J,Haddad N I A,Yang S Z,Mu B Z.Applied Biochemistry and Biotechnology,2010,160(3):812.