NJCU - Works of Cell Biology

Sunday, December 23, 2007

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3 Comments:

At January 17, 2008 1:50 PM, Anonymous Britt said...

Dr. Arrigo, Is wonderful.

 
At February 03, 2008 4:23 AM, Anonymous Anonymous said...

An Enzyme Kinetic Study
Lenin Matute, Department of Biology, New Jersey City University

ABSTRACT Enzymes are utilized to speed up biochemical reactions that would otherwise occur too slowly to be consistent with life. These biocatalysts are proteins that possess a binding site where reactant molecules, substrates, will fit. They play a diversified role in everyday life including the production of food and its digestion and have found several ex-vivo industrial applications. Here, the kinetic parameters of Catecholase from Solanum tuberosum were determined. The enzyme activity was assessed along several relevant parameters with the spectrophotometrically measurable colored product, benzoquinone used as an indicator of enzyme activity. Results indicate that reaction rate was increased as a function of enzyme concentration. Kinetic results allowed Vmax and Km and to be estimated (0.13 Absorbance units/min and 1.75 concentration units respectively) and determined by the double reciprocal plot (0.5 absorbance units/min and 12.5 concentration units respectively). This work supports the work of others and shows that Solanum tuberosum Catecholase can be partially characterized using spectroscopy.

RESULTS
A. Effect of enzyme concentration on enzyme activity
Solanum tuberosum cell extract containing Catacholase, a pH 7 buffer solution, and ectopic catechol were utilized for the enzyme kinetic assay. A series of reaction mixtures were prepared where substrate concentration was uniform and enzyme concentration was varied. After the reactions were initiated by addition of substrate any spectrophometrically relevant product that absorbed at 420nm was measured as a function of time. As indicated in Table 1 absorbance readings were taken at 0, 2, 4 and 6 minutes. Absorbance as a function of time was plotted for each sample (Figures 1A-C, supplementary section) and the initial rates for each reaction were calculated (Table 2). Only initial rates were considered here since several systems showed the suggestion of saturation at later times.

Table 1 Effect of Enzyme Concentration on Enzyme Activity
Absorbance (420nm)
minutes
0 125 250 375 500
0 0 0.05 0.11 0.14 0.44
2 0.01 0.40 0.45 0.64 0.95
4 0.01 0.61 0.66 0.95 1.0
6 0.01 0.69 0.85 0.96 0.95

Table 2 Effect of Enzyme Concentration on the Reaction Rate
The reaction rate resulted from the slope of each reaction curve.
Enzyme
quantity Reaction
Rate (Abs/min)
0 0.05
125 0.175
250 0.170
375 0.25
500 0.255


B. Effect of substrate concentration on enzyme activity
Solanum tuberosum cell extract containing Catacholase, pH 7 buffer solutions, and ectopic catechol were utilized for another enzyme kinetic assay. A series of reaction mixtures were prepared where enzyme concentration was uniform and substrate concentration was varied. After the reactions were initiated by addition of enzyme any spectrophometrically relevant product that absorbed at 420nm was measured as a function of time. As indicated in Table 3, 1X, 2X, and 4X enzyme were analyzed and the resulting activities were recorded. Initial velocity was established for each condition (Figure 2 and Table 4). A plot of Vo as a function of [S] (Figure 3) allowed Vmax and Km to be estimated at 0.13/minute and Km = 1.75 respectively. The reciprocal of these values were plotted to determine the value of Vmax and Km at 0.5 Absorbance units /minute and 12.5 units respectively (Figure 4).


Table 3 Effect of Substrate Concentration on Reaction Rate (velocity)
Absorbance (420nm)
Tube
0min 2minutes 4minutes 6minutes
1X 0.02 0.10 0.115 0.215
2X 0.07 0.22 0.34 0.41
4X 0.09 0.35 0.54 0.66

Figure 2 legend: Using the first segments (0-2min) for each line, the initial velocity was calculated. The slope of line m is equal to Vo.

The slope calculated from each reaction curve in Figure 2 is equals to the initial reaction velocity Vo. See Table 4.
Table 4 Effect of Substrate Concentration on Initial Rate (Vo)

Tubes Vo
None 0
1X 0.04
2X 0.075
4X 0.13

A graph was derived from Table 4 to estimate Km and the Vmax of the enzymes. This graph is a plot of Vo vs. substrate concentration [S]. See Figure 3 where the horizontal line signifies ½ Vmax and the arrow points to the estimated value of Km.
The reciprocal of these values were plotted to determine the value of Vmax and Km (Figure 4).

Determining Km and Vmax for Catecholase

Figure 3 legend: When initial velocities are plotted as a function of substrate concentration Vmax and Km can be estimated. Vmax is the highest velocity indicated and Km is the [S] at ½ Vmax. This estimate indicates Vmax = 0.13/minute and Km = 1.75. Km and Vmax determined from 1/vo vs 1/[S]

Figure 4 legend: When the reciprocal of initial velocities are plotted as a function of the reciprocal of substrate concentration the double reciprocal plot allows Vmax and Km to be determined. Vmax is obtained by consideration of where the line crosses the Y axis, 1/Vmax= Y intercept; and Km is obtained by consideration of where the line crosses the X axis, -1/Km= X intercept. Vmax and Km are determined as 0.5/minute and 12.5 respectively. (On the x-axis the line crosses at -0.08).

Discussion
This investigation revealed several parameters of the enzyme Catecholase from Solanum tuberosum. It is noted that the lower rates of reaction observed at higher time points in the higher enzyme concentration samples suggest either substrate depletion or product inhibition. The former interpretation is favored by Douka’s study of 1980 which shows that under specialized reaction conditions the reaction velocity is directly proportional to cell extract and at longer reaction times the enzyme activity reaches a plateau because the substrate concentration becomes limiting. This work with Solanum tuberosum cell extract also suggests that the leveling off of reaction rates observed in part B where substrate is titrated might have been caused by either saturation of enzyme binding site or depletion of substrate. Since saturation was observed at substrate concentrations that were suspected to be limiting in the initial study on the effects of enzyme concentration, it may also indicate that there was a high enzyme affinity to the substrate so that it quickly achieved its maximum rate of reaction, Vmax.
It is suggested that enzymes affinity to substrates dictates how fast an enzyme can achieve the maximum reaction rate. Enzymes with greater affinity to substrates can result in rapidly achieving the maximum reaction rate as concentrations of substrate increase. If this affinity of enzymes to substrate was low, the enzyme is suspected to need higher concentrations of substrate to reach Vmax.

REFERENCES
Douka, C. Kinetics of Manganese Oxidation by Cell-Free Extracts of Bacteria Isolated from Manganese Concretions from Soil. Appl. Environ. Microbiol. 1980 January; 39(1): 74-80

-Lenin Matute

 
At February 23, 2008 12:42 PM, Anonymous Anonymous said...

Instrument Development and Utilization for a Quantitative Study

Patricio Fuentes, Department of Biology, New Jersey City University


Abstract: Spectrophotometry has a number of applications in the field of science. In the medical field, spectroscopy is often used to determine the presence of protein in the cerebrospinal fluid. The spectrophotometer provides helpful information to clinicians in obtaining a differential diagnosis for a specific pathology. Here, spectrophotometry was employed to develop a standard curve instrument and attempt to determine the concentration of an unknown protein sample. Results indicate that the linear portion of the developed curve instrument fell between 0.25mg/ml and 1.0 mg/ml. Absorbance of the unknown sample fell below the lowest value of the linear range and therefore could not be determined in the expected fashion. Findings with respect to the standard curve instrument support Beer’s Law where concentration is a function of absorbance.

Results:

Development of a standard curve instrument
A twofold serial dilution from a 4mg/mL stock solution of Bovine Serum Albumin was implemented to develop different protein concentrations that ranged from 2mg/mL to 0.125mg/ml. The diluent used was deionized water, and 150 ul of the diluent was used in each dilution. A dye reagent was used in accordance with the Bio-rad specifications. Calibration of the spec-20 was done prior to use. Results obtained from the spectrophotometer revealed the linear relationship of the standard instrument curve to be between 0.25mg/ml and 1 mg/ml. Graphical interpretation of the results and numerical data (Figure 2) are presented.

Determination of the protein concentration of an unknown solution
Unknown “B” was examined for its absorbance in the presence of the dye reagent. The absorbance observed resulted to be below the standard curve instrument obtained. Protein concentration was unable to be determined from the selected unknown. Error may have occurred while using the pipettor to retrieve the 100 l of the unknown solution.


Discussion:
There are many tools, like the microscope, available to scientists and researchers that facilitate in deriving qualitative and quantitative results. These tools, while important, have limited ability in the measurement of molecules. More present-day instruments like the spectrophotometer have opened up new doors for clinicians and researchers alike. Throughout this investigation, the ideas of Beer and Lambert were revealed and applied, as the linear relationship of the developed standard curve would reveal the absorbance of the unknown protein sample. The implementation of spectrophotometry can be found in the work of Lowry et al (1951). In his investigation, protein is measured with the folin phenol reagent. Vieira et al (1992) go on to compare Lowry’s method and the Coomassie blue method for the determination of protein concentration. In their investigation, a standard curve instrument was developed, and all photometric data were obtained from reaction mixtures of BSA and test solutions. These researchers and others have paved the way for future scientist to determine protein quantification in the scientific setting, as well as, the clinical setting.
References:
1) Lowry O.H, Rosebrough N, Lewis F A, Randall R (1951). Protein Measurement with the Folin Phenol Reagent. Journal of Biochemistry, (193): 265-275.
2) Vieira EG, Henriques SB (1992). Comparison of the Lowry and Coomassie Blue methods for the determination of protein concentration. Brazilian Journal of Medical and Biological Research, 25(6):583-91.

 

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