The analysis of creatine phosphokinase isoenzymes using the luciferin-luciferase system

Abstract

The study is concerned with the electrophoretic methods of analysis of creatine phosphokinase isoenzymes using the luciferin-luciferase detection system. A statistical analysis of the results of total creatine phosphokinase levels using a modified Rosalki method and the luciferin-luciferase system was undertaken and showed very good correlation between the two methods. This work determined the conditions necessary for detectable quantities of light to be emitted from electrophoresed cellulose acetate plates. It was found that by decreasing the amount of water needed to reconstitute the luciferin-luciferase kit greater values of emitted light were observed. It was also found that the developing cellulose acetate plate should not be sandwiched between glass plates since this will greatly reduce light emission. It was also discovered that adenosine monophosphate should be present in very small quantities or not at all since it inhibits the luciferin-luciferase reaction. Once the problem of obtaining adequate luminescence was overcome this analysis turned to electrophoresis. The original procedure gavenoff an adequate amount of luminescence but diffusion of the proteins on the wet developing cellulose acetate plate did not allow detection of the separated creatine phosphokinase isoenzymes. This problem was reduced to a minimal amount by allowing the adenosine triphosphate to accumulate while the developing plate was sandwiched between two glass plates. This limited the amount of diffusion which occurred. Detection with a standard fluorometer with the UV light source blocked off gave adequate results. Detection was also attempted using photographic plates but none of the plates were detectably exposed by the small quantities of light present. This work, therefore, indicated that analysis of CPK isoenzymas using the Luciferin-Luciferase System (LLS) technique is feasible. The LLS method therefore probably can be used to determine with a high degree of accuracy if a patient with an elevated total creatine phosphokinase level suspected of having undergone a myocardial infarction had indeed suffered such an attack.