CN1289688C - 总半胱氨酸测定 - Google Patents
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Abstract
本发明提供一种测定生物液体中总半胱氨酸的方法，该方法使用高半胱氨酸酶和非特异性脱硫酶类似处理的所述液体部分。
Description
相关申请的交叉参考
本申请依据35U.S.C.§119而要求2001年11月20日递交的临时申请60/333532的优先权。该申请内容在此引入作为参考。
技术领域
本发明涉及血或血浆中总半胱氨酸的酶法测定。
背景技术
血浆中总半胱氨酸含量(tCys)已经明显成为评价心血管疾病风险的重要参数。有报告说心血管疾病风险低与在250-275μM的tCys水平相关，而风险较高则与较低水平(＜225μM)和较高水平(＞300μM)都相关。El-Khairy，等Circulation(2001)2544-2549。作为半胱氨酸同系物的高半胱氨酸的水平(tHcy)也与之相关，因为在高半胱氨酸水平高时静脉血栓形成和心肌梗塞的风险升高。Marcucci，等，M J.Clin.Pathol.(2001)116：56-60。因为半胱氨酸和白蛋白是循环中高半胱氨酸的共价载体，并且半胱氨酸也是高半胱氨酸细胞摄取的竞争者和经由转硫作用途径的高半胱氨酸代谢物，所以半胱氨酸和高半胱氨酸的水平是相关的。Hortin，等J.Clin.Chem.(2001)47：1121-1124。
另外还已知，在由于转硫作用途径的缺陷而导致高半胱氨酸水平很高的个体中，总半胱氨酸水平低。据估计人群中有百分之一在酶负责(enzymeresponsible)中有缺陷；纯合性缺陷导致高半胱氨酸尿症。tCys/tHcy比例有助于鉴定杂合子(heterozygotes)。Boddie，等，Metabolism(1998)47：207-211。
另外，总半胱氨酸水平与年龄，总胆固醇浓度，舒张压和咖啡摄入量相关，但与高半胱氨酸水平不同的是，与吸烟情况，叶酸盐和维生素摄入，心率和身体活动无关。El-Khairy，等，Am.J.Clin.Nutr.(1999)70：1016-1024。tCys和tHcy都与肾衰相关。Mansoor，等Clin.Chem.(1993)39：980-985。
用酶法测定总高半胱氨酸的方法在，例如，2002年10月22日授权的美国专利6,468,762和美国专利6,066,467；5,998,191；和5,985,540中已述，其公开在此引入作为参考。
另一种测定总半胱氨酸的方法也在上述‘762专利中描述，其中待测样品用非特异性脱硫酶处理。为直接测定半胱氨酸，将S-腺苷高半胱氨酸水解酶(SAHH)和腺苷加入含非特异性脱硫酶的样品，其中所述SAHH的存在量足以催化所述样品中所有的高半胱氨酸被转化为SAH，从而保护其免受脱硫酶作用。因此，测量的仅为样品中半胱氨酸的水平。
本发明提供了基于酶来测定总半胱氨酸的替代测定法。两种测定法的存在使得不仅可以分别测定总高半胱氨酸和总半胱氨酸，而且可测定tCys/tHcy的比例。
发明内容
本发明目的是确定生物液体，优选血浆中的总半胱氨酸，伴有或者可控确定总高半胱氨酸以及高半胱氨酸和半胱氨酸的浓度之和。通过利用，在一个反应器中与高半胱氨酸和半胱氨酸反应而产生氨，硫化氢，及各自的α-酮羧酸(α-酮戊二酸或丙酮酸)的酶，及在另一个反应器中仅与高半胱氨酸进行这种脱硫酶(裂解酶)反应的酶，根据浓度差异可以确定总半胱氨酸水平。所述结果在血浆中2μM-1,000μM的范围内呈线性。
因而，一方面，本发明涉及一种测定生物液体中总半胱氨酸浓度(tCys)的方法，该方法包括
a)用脱硫酶处理所述液体的第一样品(该酶以高半胱氨酸和半胱氨酸作为底物)，并测量所述脱硫酶至少一种产物的水平，从而确定半胱氨酸(tCys)和高半胱氨酸(tHcy)的浓度之和；
b)用脱硫酶处理所述生物液体的第二样品(该酶特异以高半胱氨酸作为底物)，并测量至少一种产物的水平，从而确定高半胱氨酸(tHcy)的总浓度；并且
c)从步骤a)得到的tCys加tHcy中减去步骤b)得到的tHcy，获得总半胱氨酸浓度。
另一方面，本发明涉及一种测定总半胱氨酸浓度的试剂盒，它包括，在合适的容器中，以半胱氨酸和高半胱氨酸作为底物的脱硫酶，特异以高半胱氨酸作为底物的脱硫酶，和可选的，有效还原二硫键的还原剂，及可选的，检测至少一种产物的试剂，及进行此测定的说明书。
附图说明
图1的图显示了本发明所述测定的典型标准曲线。
具体实施方案
本发明利用脱硫酶的特异性特性来确定生物液体中的高半胱氨酸和半胱氨酸水平。合适的生物液体包括，例如，尿，脑脊液，血液和血浆或血清。大多数关于这些水平的显著意义的文献涉及血浆或血清。通常需要在进行测定前用有效还原二硫键的还原剂(如二硫赤藓糖醇)处理样品，从而将半胱氨酸和高半胱氨酸从它们的巯基形式释放，以便实施脱硫酶作用。
本发明所用的脱硫酶将高半胱氨酸转变为α-酮戊二酸，氨和硫化氢并可以将半胱氨酸转变为丙酮酸，氨和硫化氢。所述测定可包括测定这些产物中的任一种。但测定氨或硫化氢会更方便，因为可以对这两种氨基酸的产物采用相同的方法。特别优选测定硫化氢，因为此测定方法相对容易。然而，本领域已知的测定α-酮戊二酸，丙酮酸，氨的方法和测定这些产物水平的方法也可应用。
一种方便测定硫化氢生成水平的方法见上述美国专利6,468,762。简言之，此方法包括含氧化剂如铁氰化钾的第一种产色试剂，和含N，N-二烷基苯二胺的第二种产色试剂。这些试剂与所得的硫化氢反应形成有色的复合物，该复合物可用吸光度进行分光光度测定，或用所述复合物的荧光进行更灵敏的测定。优选的二烷基苯二胺是N，N-二丙基苯二胺(DPPDA)或N，N-二丁基苯二胺(DBPDA)。
然而，可用任何测定所述反应产物的方法。
如上述，所述的脱硫酶之一会以半胱氨酸和高半胱氨酸作为底物，从而当用此脱硫酶处理生物液体一段适宜的反应时间，如10分钟或20分钟后，所述脱硫酶将会把生物样品中的高半胱氨酸和半胱氨酸都转变为上述产物。此酶适合从微生物制备。一个实例为从恶臭假单胞菌(Psedomonas putida)重组制备的脱硫酶。此酶为Tan，等，Protein Purification & Expression(1997)9：233-245所述。
本文中称为“高半胱氨酸酶(homocysteinase)”的高半胱氨酸特异性脱硫酶被限定为，以高半胱氨酸作为底物的脱硫酶活性优于以半胱氨酸作为底物的活性，导致用所述酶处理受试者血液，尿，组织液，血清，或血浆样品时所释放的硫化氢量基本上是从所述样品中的高半胱氨酸产生而不是从半胱氨酸产生，甚至当所述样品中高半胱氨酸浓度比半胱氨酸浓度小10倍时也是如此。或者，所述高半胱氨酸酶具有如下性质：当所述液体中高半胱氨酸和半胱氨酸的浓度分别为大约5-15μM和大约100-300μM时，由所述高半胱氨酸酶接触生物液体而产生的硫化氢至少约90％是由高半胱氨酸产生的，或者，当所述液体含5μM高半胱氨酸1,000μM半胱氨酸时，由所述高半胱氨酸酶接触生物液体而产生的硫化氢至少大约90％是由高半胱氨酸产生的。此类高半胱氨酸酶可参见上述专利；及Han，等Protein Expression &Purification(1998)14：267-274。
如上述，可将生物液体分为两份样品，优选对它们进行相同处理。通常，将脱硫酶加到样品中使终浓度达到0.01-1,000μg/ml；并加入试剂，得到终浓度为1-100mM，更优选5-50mM的缓冲液；0.01-100mM，甚更优选0.1-10mM的二硫键还原剂。剩余试剂的性质依赖于该测定所选的检测系统。
在一特别优选的检测方法中，通过测定所产生的硫化氢的浓度或量来确定产物水平。特别优选的是形成具有荧光性质的生色团。所述能够吸收光并发射荧光的复合物，通过与N，N-二烷基苯二胺，优选N，N-二丙基苯二胺(DPPDA)或N，N-二丁基苯二胺(DBPDA)反应得到。之后用合适的氧化剂(例如铁氰化物形式的铁离子)来氧化所述复合物。带有颜色的试剂的水平可以通过通常在约670-680nm测量吸光度来进行测量，而此复合物的荧光可以利用约665nm或640nm的激发波长、并在较短波长测量相应发射来进行测量。荧光检测对比于(as opposed to)吸光度的应用如下所述，其显示可提高所述测定的灵敏度。
含非特异性脱硫酶的样品的结果提供了tHcy加tCys的浓度水平。含高半胱氨酸酶的样品的结果提供了tHcy浓度。因此，这些浓度之差代表了生物液体中的总半胱氨酸浓度。
下述实施例是为阐述而不是为了限定本发明。
实施例1
测定半胱氨酸和高半胱氨酸
将待测样品分为两部分：一部分用非特异性脱硫酶处理而另一部分用高半胱氨酸酶处理。
各例中，将10-20μl的样品及相应的980-990μl转变(Conversion)缓冲液混合，得到总共1ml的样品，将该样品置于37℃保温30分钟。所述转变缓冲液为20mM的磷酸钾，pH8.3，150mM NaCl，30μg/ml，0.2％Triton X-100，和1mM DTT。
然后用10μl重组高半胱氨酸酶或非特异性脱硫酶(0.275mg)处理所述样品。所述脱硫酶为重组生产并如Tan，等(见上文)所述从恶臭假单胞菌分离。所述高半胱氨酸酶见Han，等(见上文)所述。两份样品都在37℃保温10分钟。
然后通过加入50μl色原体(6M盐酸中的40mM N，N-二丁基-苯二胺盐酸)终止该反应，随后加入50μl氧化剂(20mM磷酸钾，pH8.3中40mM的铁氰化钾)。然后在37℃保温所述部分10分钟并通过用激发波长665nm和发射波长690nm的荧光或者675nm的吸光度来读数。
Claims (10)
1.确定生物液体中总半胱氨酸浓度的方法，该方法包括
a)用以高半胱氨酸和半胱氨酸作为底物的非特异性脱硫酶处理所述液体的第一样品，并测量所述脱硫酶的至少一种产物的水平，以此确定总半胱氨酸和总高半胱氨酸的浓度之和；
b)用特异以高半胱氨酸作为底物的高半胱氨酸酶处理所述液体的第二样品，并测量至少一种产物的水平，从而确定总高半胱氨酸的浓度；并且
c)从步骤a)所得的总半胱氨酸浓度和总高半胱氨酸浓度之和中减去步骤b)所得总高半胱氨酸浓度，从而获得总半胱氨酸浓度。
2.权利要求1所述的方法，其中在步骤a)和步骤b)中测量的所述产物是硫化氢。
3.权利要求2所述的方法，其中所述硫化氢通过用氧化剂和二烷基苯二胺处理来测量。
4.权利要求3所述的方法，其中所述氧化剂是铁离子。
5.权利要求1所述的方法，其中所述非特异性脱硫酶分离自恶臭假单胞菌。
6.权利要求1所述的方法，其中所述高半胱氨酸酶分离自T.vaginalis。
7.权利要求1所述的方法，其中所述生物液体为血清或血浆。
8.用于测定总半胱氨酸浓度的试剂盒，它在合适的容器中包含以半胱氨酸和高半胱氨酸作为底物的非特异性脱硫酶，特异以高半胱氨酸作为底物的高半胱氨酸酶。
9.权利要求8所述的试剂盒，它还包含有效还原二硫键的还原剂。
10.权利要求8所述的试剂盒，它还包含检测至少一种产物的试剂。
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| US9200251B1 (en) | 2011-03-31 | 2015-12-01 | David Gordon Bermudes | Bacterial methionine analogue and methionine synthesis inhibitor anticancer, antiinfective and coronary heart disease protective microcins and methods of treatment therewith |
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