Ripon College1, Chemistry, Ripon, WI 54971 Medical College of Wisconsin2, Biophysics, Milwaukee, WI 53226
Michaelis-Menten constants (Km’s) represent the dissociation constants of specific enzymes. Due to the nature of enzymes and the fact that they are almost always less concentrated than their specified substrates, an enzymatic reaction will reach a maximum rate or velocity when all enzyme particles are complexed with substrate. At that point, the reaction can only proceed as fast as the product particles can detach. The idea of Vmax describes the maximum speed a certain reaction can possibly go at a given concentration, when all reaction sites are in use, and the Km is the substrate concentration at half the Vmax. When an enzymatic reaction follows Michaelis-Menten kinetics, the rate increases steeply at lower concentrations but begins to level out and become constant as the enzyme becomes saturated; a graph of concentration versus rate would take the form of a rectangular hyperbola.
L-Cysteine is an amino acid with a thiol group that can be modified to form S-nitroso-L-cysteine, or Cys-NO. Cys-NO stops respiration in cells and turns critical intracellular thiols into intracellular -SNO’s (S-nitrosothiols), and at concentrations higher than 500µM, eventually kills the cells. It is known to break down in light, but was also found to break down even more quickly when left at room temperature or with exposure to any amount of metal ions. For this reason, all reactions with Cys-NO need to be done in the presence of DTPA (a metal chelator) and any samples have to be kept on ice. This compound enters cells through an L-amino acid transport (L-AT) protein, however, the Km and Vmax are not known. The objective of this research was to find them, which would also prove the enzymatic transport mechanism. By measuring the rate of uptake over a given time period at varying concentrations of Cys-NO and then plotting the rates versus the starting concentrations, the Km and Vmax can be extrapolated. To measure the rate, the extracellular concentration of Cys-NO can be measured at consecutive intervals and plotted versus time. The concentration of Cys-NO can be tested in several ways. The first is the Griess-Saville reaction which measures the total nitrite level and then the total –SNO level, with the difference between the two being the Cys-NO concentration; however, this proved relatively inaccurate. The second and presumably the best method of measuring Cys-NO concentration is to measure the absorbance at 334nm with a spectrophotometer. As the extinction coefficient is 0.9 for a 1mM solution, the concentration can quite easily be calculated using Beer’s law (A=εbc). This method is clear and accurate until about 25µM, wherein the data starts to get rather noisy and the instrument reaches a sensitivity threshold. The final method, which would be useful for concentrations under what the spectrophotometer was capable of measuring, was the use of chemiluminescence.
To get an idea for the time frame of this reaction, the uptake was first tested in twenty minute intervals over an eighty minute time frame, over RAW cells. Samples were taken in triplicate and put directly onto ice. The uptake leveled out after about forty minutes, so the time frame for testing was adjusted, with samples taken every five minutes for forty minutes. Concentrations were tested both above and below 50µM to determine relative placement on the curve, and from these it was determined that the rate was still climbing. Assays were done at concentrations up to 1mM, however, any rate taken above 500µM was unreliable due to the number of cells that were dying, which happened sooner and sooner as the concentration was increased. Even with adjustments to the time frame, it soon became close to impossible to obtain a confident rate at all.
When the rates that were reliable were graphed against their starting concentrations, a best-fit Km and Vmax were found, however, it is quite possible that an entirely correct Km and Vmax cannot be found because of the detrimental effects of Cys-NO on a cell’s ability to survive. Though there is plenty of data to support the lower end of the curve, the higher saturation points are not well represented in the data, and so the more influential part of the curve might be different than what is proposed by these data.
[Abstract (DOC)]