Abstract
A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography (RP-HPLC). The activity of hexokinase was determined by detecting the amount of ADP in the reaction that converts glucose into glucose-6-phosphate. The separation degree of ATP and ADP was as good as 3.46 (separation degree>1.5). In the range of 0.01–0.40 mmol/L, there was a good linear relationship between concentration of ADP and its peak areas (the correlation coefficient was 0.999). The relative standard deviation(RSD) of intraday was 1.43%–1.46%, that of interday was 2.12%–2.15%. At 30 °C (optimal temperature) and pH7.0 (optimal pH), the recovery rate of hexokinase was 95.3%–97.6%. The results showed that the method had good precision and high recovery rate, and the enzyme activity was determined through only one-step reaction. To some extent, this method can reduce the cost.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Wang Jingyan, Zhu Shenggeng, Xu Changfa. Biochemistry [M]. Beijing: Higher Education Press, 2002(Ch).
Heidi C D, Todd A L, Jeremy W C, et al. Quercetin, a fluorescent bioflavonoid, inhibits Trypanosoma brucei hexokinaseI[J]. Experimental Parasitology, 2011, 127: 423–428.
Muthuraman P, Srikumar K. Induction of hexokinase I expression in normal and diabetic rats by a brassinosteroid isoform [J]. European Journal of Pharmaceutical Sciences, 2010, 41: 1–9.
Tiago C L, Raquel G C, Daniel D S. Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice [J]. FEBS Letters, 2011, 585: 92–98.
Baque S, Montell E, Guinovart J J, et al. Expression of glucokinase in cultured human muscle cells confers insulin-independent and glucose concentration-dependent increase in glucose disposal and storage [J]. Diabetes, 1998, 47(9): 1392–1398.
Wu Gang, Zhao Yingchao, Cheng Jing, et al. Expression and clinical significance of erythrocytic hexokinase in patients with non-small cell lung cancer [J]. Chinese Clinical Oncology, 2007, 12(2): 91–93(Ch).
Katsanidis E, Addis P B. Novel HPLC analysis of tocopherols, tocotrienols, and cholesterol in tissue [J]. Free Radical Biology and Medicine, 1999, 27: 1137–1140.
Wang Jingzhu, Yan Runan, Guan Hui, et al. HPLC determination astragaloside content in astragalus processed products [J]. Chinese Medicine, 1998, 23(3): 84–89(Ch).
Jin Dongyue. The Applications of inversed phase chromatography in environmental analysis [J]. Sun Yatsen University Forum, 2002, 22(5): 1–6.
Dietmar E B. Simultaneous HPLC determination of carotenoids used as food coloring additives: Applicability of accelerated solvent extraction [J]. Food Chemistry, 2004, 86(3): 449–456.
Sarowha S L S, Sharma B K, Sharma C D, et al. Bhagat characterization of petroleum heavy distillates using HPLC and spectroscopic methods [J]. Energy Fuels, 1997, 11(3): 566–569.
Faria A M, Magalhães D R, Collins K E, et al. Characterization of zirconized silica supports for HPLC [J]. Analytica Chimica Acta, 2005, 550(2): 137–143.
Liu Guiyang, Mao Xiangju, Chen Jinyang. The fundamental principle and application of reversed-phase high-performance liquid chromatography [J]. Ningbo Chemical Industry, 2008, 22–25(Ch).
Veciana-Nogues M T, Izquierdo-Pulido M, Vidal-Carou M C. Determination of ATP related compounds in fresh and canned tuna fish by HPLC [J]. Food Chemistry, 1997, 59(3): 467–472.
Kochanowski N, Blanchard F, Cacan R. Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC [J]. Analytical Biochemistry, 2006, 348: 243–251.
Junichi S, Jun T, Hiroaki I, et al. Assay of ATPase and Na, K-ATPase activity using high-performance liquid chromatographic determination of ADP derived from ATP [J]. Journal of Chromatography B, 2000, 744: 19–23.
Miao Yu, Wang Chenglong, Yin Huijun. High performance liquid chromatogram (HPLC) determination of adenosine phosphates in rat myocardium [J]. Journal of Peking University (Health Sciences), 2005, 37(2): 201–202(Ch).
Wang Yuehao. Experimental Guide of Modern Plant Physiology [M]. Beijing: Science Press, 2004(Ch).
Patrick K A, Luigi J A, Dayie T K. Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway [J]. Protein Expression and Purification, 2011, 76: 229–237.
Volonté M G, Yuln G, Quiroga P, et al. Development of an HPLC method for determination of metabolic compounds in myocardial tissue [J]. Journal of Pharmaceutical and Biomedical Analysis, 2004, 35: 647–653.
Liu Junze, Gao Wenxiang, Cao Lifei, et al. Changes of adenylate pool and energy charge in mitochondria isolated from rat brain exposed to hypobaric hypoxia [J]. Acta Academiae Medicinae Militaris Tertiae, 2003, 25(24): 2165–2168(Ch).
Ma Chunyu, Duan Yu. Progress of glucokinase and type 2 diabetes [J]. Foreign Medical Sciences, 2003, 24(6): 318–319(Ch).
Author information
Authors and Affiliations
Corresponding author
Additional information
Foundation item: Supported by the Key Project of Science and Technology Department of Fujian Province (2008Y0052)
Biography: GUAN Yuxia, female, Ph. D., research direction: microbial pharmaceutics.
Rights and permissions
About this article
Cite this article
Guan, Y., Wang, J. & Sun, J. A method for determination of hexokinase activity by RP-HPLC. Wuhan Univ. J. Nat. Sci. 16, 535–540 (2011). https://doi.org/10.1007/s11859-011-0793-0
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11859-011-0793-0