High-performance liquid chromatographic determination of creatine kinase activity influenced by methylglyoxal.
Protein glycation has been implicated in the development of diabetic complications and other health disorders, which mainly arise from accumulation of advanced glycation endproducts (AGEs) in vivo. Methylglyoxal (MGO), a typical reactive intermediate carbonyl formed in early glycation process, can react non-enzymatically with N-terminal amino groups on proteins, leading to their inactivation and generation of detrimental AGEs. Recently, it was reported that activity of creatine kinase (CK, EC 2.7.3.2) could be reduced or even eliminated completely after incubation with MGO in vitro. CK activity is usually determined by conventional colorimetric assays. However, these methods are not appropriate for monitoring the influence of MGO on CK activity since MGO can also directly react with creatine, a substrate of CK. In this study, an efficient and much more accurate HPLC approach was established to investigate the effect of MGO on CK activity. Aminoguanidine was utilized to eliminate interference from the undesirable reaction between residual MGO and creatine. It was found that higher concentrations of MGO and longer incubation time for CK and MGO caused more pronounced reduction in CK activity. This HPLC method greatly facilitates acquisition of kinetic data about CK reaction and through further improvement it may be adopted to rapidly screen potential inhibitors of MGO-induced glycation.
Peng
Xiaofang
X
School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, People's Republic of China.
Ma
Jinyu
J
Cheng
Ka-Wing
KW
Chen
Bo
B
Chen
Feng
F
Wang
Mingfu
M
D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003402:Creatine Kinase / Q000378:metabolism*; D003627:Data Interpretation, Statistical; D006146:Guanidines / Q000737:chemistry; D009132:Muscles / Q000737:chemistry; D011765:Pyruvaldehyde / Q000494:pharmacology*; D011817:Rabbits
D006146:Guanidines; D000244:Adenosine Diphosphate; D011765:Pyruvaldehyde; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; C004479:pimagedine