基于棉纤维素纳米晶体的荧光肽作为弹性蛋白酶传感器的动力学和结构分析。

Kinetic and structural analysis of fluorescent peptides on cotton cellulose nanocrystals as elastase sensors.

机构信息

USDA-ARS, Southern Regional Research Center, Cotton Chemistry & Utilization, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, United States.

出版信息

Carbohydr Polym. 2015 Feb 13;116:278-85. doi: 10.1016/j.carbpol.2014.04.067. Epub 2014 Apr 26.

DOI:10.1016/j.carbpol.2014.04.067

PMID:25458301

Abstract

Human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) are serine proteases with destructive proteolytic activity. Because of this activity, there is considerable interest in elastase sensors. Herein we report the synthesis, characterization, and kinetic profiles of tri- and tetrapeptide substrates of elastase as glycine-esterified fluorescent analogs of cotton cellulose nanocrystals (CCN). The degree of substitution of peptide incorporated in CCN was 3-4 peptides per 100 anhydroglucose units. Glycine and peptide-cellulose-nanocrystals revealed crystallinity indices of 79 and 76%, respectively, and a crystallite size of 58.5 Å. A crystallite model of the peptide-cellulose conjugate is shown. The tripeptide conjugate of CCN demonstrated five-fold greater efficiency in HNE than the tripeptide in solution judged by its kcat/Km of 33,515. The sensor limits of detection at 2mg of the tri- and tetrapeptide CCN conjugates over a 10 min reaction time course were 0.03 U/mL PPE and 0.05 U/mL HNE, respectively.

摘要

人中性粒细胞弹性蛋白酶（HNE）和猪胰弹性蛋白酶（PPE）是具有破坏性蛋白水解活性的丝氨酸蛋白酶。由于这种活性，弹性蛋白酶传感器引起了相当大的兴趣。在此，我们报告了弹性酶三肽和四肽底物的合成、表征和动力学谱，这些底物是弹性酶的甘氨酸酯化荧光类似物，是棉纤维素纳米晶体（CCN）的类似物。肽在 CCN 中结合的取代度为每 100 个脱水葡萄糖单位 3-4 个肽。甘氨酸和肽-纤维素纳米晶体的结晶度指数分别为 79%和 76%，结晶尺寸为 58.5Å。显示了肽-纤维素缀合物的晶体模型。与溶液中的三肽相比，CCN 的三肽缀合物在 HNE 中的效率高五倍，其 kcat/Km 为 33515。在 10 分钟的反应时间内，三肽和四肽 CCN 缀合物的传感器检测限分别为 0.03 U/mL PPE 和 0.05 U/mL HNE。