Lactose Assay using Spectrophotometry to detect and analyze Competitive Inhibition from ONPG

By: Melissa Manetsch and Maggie Shutts Abstract: The purpose of this research is to determine if it is possible to develop a qualitative assay to assess whether a sample containing an unknown concentration of lactose is below or above the threshold of an individual’s tolerance. Our research would then be able to determine if it is valid to create an assay in which quantifying the effects of varying concentrations of lactose on ONPG due to competitive inhibition may be compared in order to qualitatively classify lactose concentrations (ONPG is a molecule consisting of a galactose and ortho-nitrophenol dye monomer, which when hydrolyzed by lactase releases the yellow dye). This was determined through research to determine the correlation is between lactose concentration the initial rate of reaction of the lactase enzyme. Verifying this would mean that the rate of reaction of ONPG in solution of known concentration of lactose can be compared to that in a solution of unknown concentration of lactose to determine whether the unknown sample contains a concentration of lactose either below or above the known amount. Ultimately, the goal is to develop a simple lactose measurement device. The first set of methods used consisted of a series of range finding in order to determine the proper amounts of reagents. The first set of range finding was for lactase, in which ONPG was held constant and lactase was varied with a 1:10 serial dilution. It was found that the 1:100 concentration of lactase created a strong yellow color with a steady increase over 2-3 minutes. Range finding was then employed with ONPG using the 1:100 concentration of lactase and completing a 1:2 serial dilution of ONPG, with the stock solution being at maximum solubility (approximately the concentration of lactose at the cutoff concentration). Once again, it was qualitatively observed, based on the rate of production of ONP, that the first sample (maximum solubility) produced a yellow color in 2-3 minutes and that lactase would not need to be increased, as it would have the perfect rate of reaction for lactose for the same concentration. Proceeding from range finding, assay development was begun in order to help determine the adequate concentration of reagents for the final assay. The first of these experiments consisted of lactose and lactase being held constant (at the cutoff concentration and 1:100 concentration, respectively). It was found that an ONPG concentration of .0018125 M would result in an absorbance in the middle of the detection range for the spectrometer using these reagents. Moreover, a dry run of the assay was conducted in order to obtain experimental data regarding lactose concentration and rate of reaction. A 1:2 serial dilution of lactose was conducted across the dynamic range for the assay: 8% to 0.125%, as this is the approximate range of lactose in foods that, in the final product, would be tested with the assay. A distinct correlation was found between lactose concentration and resulting rate of reaction, in which there was a linear relationship between lactose concentration and rate of reaction when lactose concentration increased exponentially. Introduction: My friend Melissa has lactose intolerance, and one day she mentioned that it would be significantly more helpful for her to avoid the unpleasant and sickening symptoms of lactose intolerance if she only knew beforehand how much lactose was in her food. Upon further research, we found that approximately 65% of the human population suffers from lactose intolerance, including 90% of adults in Eastern Asia (Lactose). We then decided to assume the challenge of creating a lactose indicator, finding that it would not only help us, but also many more individuals than initially presumed. Evidently, lactose intolerance is a very prevalent dietary restriction that affects a vast majority of the population. However, the degree to which people are lactose intolerant varies from person to person. For example, some people may be able to ingest a cup of milk every few hours, while others may not even be able to ingest a small amount of butter used to cook their meal (Lactose). Because of this, the maximum amount of lactose that people can ingest without feeling symptoms varies. We will call this the “threshold of tolerance” or “cutoff concentration.” Melissa, the person for whom this specific assay is based, has a cutoff concentration of about 1%, or 0.029M lactose (Carper 2017). Our research and assay involve three prominent reagents: lactose, ONPG, and lactase. Lactose is a disaccharide sugar consisting of one glucose and one galactose molecule connected by a hydrogen bond. An ONPG (ortho-Nitrophenyl-?-galactoside) molecule is composed of a molecule of galactose and a molecule of ortho nitrophenol (ONP) connected by a hydrogen bond. Lactase is an enzyme that interacts with ONPG and lactose similarly, “hooking” onto their galactose molecules and severing their hydrogen bonds to separate the galactose from ONP and glucose, respectively. Because both ONPG and lactose provide the same substrate (a galactose molecule) for the enzyme, they act as inhibitors to one another for the active site of the enzyme. This is known as competitive inhibition, because both molecules are competing for the active site (Borralho). Our assay pivots on this concept of competitive inhibition. When the lactase hydrolyzes the ONPG molecule, it reveals a yellow dye. The concentration of dye (product) produced as a function of time can be quantified using a spectrometer by measuring the absorbance of the dye as a function of time (the spectrometer uses a 420 nm light because this is the peak absorbance of ONP). Thus, the rate of reaction of the enzyme can be determined by extrapolating this rate. However, when lactose inhibits the lactase-ONPG reaction, this slows down the rate of ONP produced. As a result, a qualitative lactose assay involving two experiments can be created using the above information. The first of which consists of a sample of constant concentrations of ONPG and lactose with lactose at the cutoff concentration. The rate of reaction is then recorded over approximately 2 minutes. The second experiment involves the same concentrations of ONPG and lactase but an unknown concentration of lactose. If the rate of reaction of ONP produced in the second experiment is smaller than that of the first experiment, then there is more inhibition and, therefore, the concentration of lactose in the unknown sample is above the cutoff concentration. If the rate of reaction of the second sample is above that of the first sample, then we know that there is less lactose inhibition and therefore the second sample contains a concentration of lactose less than then the cutoff, and is therefore safe to ingest. Objectives: The purpose of this research is determine if it is possible to develop a qualitative assay to assess whether a sample containing an unknown concentration of lactose is below or above the threshold of an individual’s tolerance. Our research would then be able to determine if it is valid to create an assay in which quantifying the effects of varying concentrations of lactose on ONPG due to competitive inhibition may be compared in order to qualitatively classify lactose concentrations. Ultimately, the goal is to develop a simple lactose measurement device. Beginning Research (Range Finding):

Lactase Range Finding

Objective: Create specified concentrations of lactase. Then, create a lactose and ONPG reaction out of the strongest concentration to produce a yellow color. Four concentrations of lactase were created using a 1:10 serial dilution, the first of which being 0.1 ml of stock solution (300 mg of lactase in 5 ml of buffer) in 0.9 ml of buffer. Four solutions of 0.05 g of ONPG in 0.35 ml of buffer were also created. Rather than using the spectrometer, we were able to determine this optimum concentration of lactase qualitatively. The 1:10 reaction occurred too fast for us to even be able to take a reference. The 1:100 created a steady increase in presence of yellow color over approximately 2 minutes before plateauing. The 1:1000 and the 1:10000 produced a very light, pale yellow, exceeding the optimal time range of 2-3 minutes as well. Constant ONPG concentration (saturated stock solution) with lactase varied.   Table 1. Approximate time until intensity of yellow color has stopped changing for the ONPG solution based on varying concentrations of lactose.

Concentration of Lactase	Approximate time until intensity of yellow color stops increasing (using visual cues)
1:10	0 (near instantaneous)
1:100	46
1:1000	2:04
1:10,000	5:32

ONPG Range Finding

Objective: Determine a concentration of ONPG that most closely matches a cutoff concentration in order to effectively determine a ratio of ONPG to lactase that creates a desired yellow color in a reasonable amount of time (approx 1-2 minutes). Below is a 1:2 serial dilution of ONPG. The first test tube consists of 2 ml of ONPG at maximum solubility (10 mg/ml). 200 ul of lactase of 1:100 concentration of lactaid pill to buffer were then added to each test tube. The resulting photo was taken after approximately 3 minutes. Converted to molarity, the farthest left test is 0.033 M, which is approximately the molarity of 1% lactose–the threshold of tolerance (0.029 M). This indicates that, because lactose and ONPG both contain galactose as their binding site, they both possess approximately the same rate of reaction for lactase under the same concentrations. Because the reaction produced a solid yellow color in the goal time for the final assay (about 2-3 minutes), it has been demonstrated that these concentrations of substrate and enzyme are in the proper range for optimal assay performance. Note: Though the previous experiment conducts lactase range finding with a concentration of ONPG that varies from the threshold, it just so happened that the concentration works sufficiently for this concentration, falling approximately just in the range–making it unnecessary for further lactase range finding. Table 2. Average rate of change of AU (absorbance units) for every 10 seconds until maximum absorbance is reached, maximum absorbance over four minutes, and time to reach maximum absorbance with a four minute maximum based on varying concentrations of ONPG in solution.

	Average rate of change of AU for every 10 seconds (until maximum AU is reached)	Maximum Absorbance (over 4 minutes)	Time to reach Maximum Absorbance (4 minute  maximum)
Sample 1 (1:2)	0.469	0.845	0:18
Sample 2 (1:4)	0.182	0.820	0:45
Sample 3 (1:8)	0.096	0.770	1:22
Sample 4 (1:16)	0.059	0.704	2:02
Sample 5 (1:32)	0.030	0.670	3:40
Sample 6 (1:64)	0.018	0.432	4:00
Sample 7 (1:128)	0.009	0.216	4:00
Sample 8 (1:256)	0.004	0.096	4:00
Sample 9 (1:512)	0.003	0.072	4:00
Sample 10 (1:1024)	0.001	0.024	4:00

Methods and Materials: Materials

• Lactose
• Ortho-Nitrophenyl-?-galactoside (ONPG)
• Lactase Enzyme
• 20 ml plastic test tubes and 10 ml glass test tubes
• 4 ml cuvettes
• Pharmacia Biotech Ultrospec 2000 Spectrophotometer

? Then, Pasco Spectrometer and spectrometer app for iPad

• 7pH Phosphate buffer

Methods I. Experiment 1: Lactase is constant (400 ul at 1:100 concentration) and ONPG is varied around 0.033M

A. The goal of this experiment was to determine a general ratio of lactase to ONPG in which the resulting absorbance after approximately 3 minutes was between 1-1.5 AU. This way, there is room for variation for absorbance when lactose inhibition results in a peak absorbance either below or above 1.5 AU. Also, the absorbance will not precede or exceed the range of the spectrometer (0-3AU). This “ideal” concentration of ONPG was found to be 0.0018125 M

II. Experiment 2: ONPG constant (0.0018125 M), lactase constant (400 ul at 1:100 concentration), and lactose varied (around cutoff concentration–1%, 0.033M)

B. The purpose of this experiment was to determine how lactose inhibition affects the rate of reaction of lactase by creating a standard curve plotting concentration of lactose and resulting rate of reaction of lactase. If there is a clear trend between these variables then the potential of a working assay can be verified.

III. Repeat previous experiment to verify assay experimentally 8 times.

A. The purpose of these experiments was to continue to verify the assay experimentally.

IV. Experiment 3: Verify assay data numerically using Michaelis Menten Equation

A. In this experiment, lactose was constant and ONPG was varied. Then, these equations were used to ensure that our dynamic range (0.25% lactose – 8% lactose) and ONPG substrate was below the concentration of substrate that reaches Vmax (the rate of reaction when the enzyme is saturated with substrate, or maximum rate of reaction). This way, comparing initial rates is a valid way of comparing concentrations of lactose based on competitive inhibition because the rates will not be constant (as they would be at Vmax).

Results: Experiment 1 Lactase is constant (400 ul at 1:100 concentration) and ONPG is varied around 0.033 M. The 1:32 concentration, or 0.0018125 M, would be the best concentration of ONPG. This is because out of all of the samples, this sample steadily increased over the optimal time range for the assay with the optimal concentration of lactase. Though each sample has a fairly close maximum absorbance and average rate of change of absorbance (see Table 1), samples 1-4 each plateaued between 1-2 minutes, making the initial rate too fast to be accurately tracked. Sample 5, however, had a constant rate for the first 3-4 minutes of reaction, making it much more reliably measured. Table 3 (from Experiment 1): Constant Lactase With Varied ONPG. Though it may appear that each sample has a similar maximum absorbance and steady increase in the average rate of change, the time to reach maximum absorbance indicates when the initial rate is no longer constant and therefore no longer able to analyzed for information about the reaction that would correlate to lactose concentration.

	Average rate of change of AU for every 10 seconds (until maximum AU is reached)	Maximum Absorbance (over 4 minutes)	Time to reach Maximum Absorbance (4:00 maximum)
Sample 1 (1:2)	0.469	0.845	0:18
Sample 2 (1:4)	0.182	0.820	0:45
Sample 3 (1:8)	0.096	0.770	1:22
Sample 4 (1:16)	0.059	0.704	2:02
Sample 5 (1:32)	0.030	0.670	3:40

Experiment 2 ONPG held constant (0.0018125 M), lactase held constant (400 ul at 1:100 concentration), and lactose varied (around cutoff concentration–1%, 0.033M) As lactose concentration increased, the resulting rate of reaction of ONPG decreased as previously predicted (Figure 1). The rates of reaction for each sample are exhibited by the slope of the absorbance over time relationship for each solution (Table 2; Table 3). Finally, by plotting the rate of reaction of each sample versus the concentration of lactose in each sample, it was clear that inhibition due to lactose resulted in a smaller initial rate. However, though the relationship appears linear, it is actually exponential. The data makes it appear like a linear relationship because lactose increases exponentially due to a 1:2 serial dilution. This means that the relationship between lactose inhibition and rate of reaction is not constant; the concentration of lactose must be squared for the rate of reaction to decrease by approximately 2 x 10^-4 AU/sec. Figure 1. Data from experiment 1 in which ONPG and lactase concentrations were held constant and lactose was varied. Above is the adjusted data exhibiting absorbance as a function of time, with each line corresponding to a different concentration of lactose. Note that though each sample may vary in the y-intercept (initial absorbance), the slope, or rate of reaction, is what is compared among samples. Table 4. Raw data exhibited in figure 2. Note the change in absorbance increases as the concentration (in molarity) of lactose in the sample (noted in the parentheses next to the sample number) decreases. Figure 2: Slopes of the individual lines of absorbance vs. time (represented above under “rate of reaction”) for the various concentrations of lactose in figure 2. Note the clear trend; as lactose increases, the rate of reaction of ONP produced decreases. Experiment 3 Verify assay data numerically using Michaelis Menten Equation (Lactase is constant and ONPG is varied) Using the adjusted Michaelis Menten Equation (Lineweaver Burke Plot) the inverse of the initial velocity (or initial rate of reaction) (1/vo) was plotted on the y-axis and the inverse of the concentration of substrate (1/[s]) was plotted on the x-axis (see figure 5). The y-intercept was then determined (8.412×10^-3) and taken the inverse of to get Vmax, or 118.878. The slope was then calculated (111.039) and then multiplied by Vmax to get Km (23836.23482 uM) and then converted to molarity to get Km of 0.0238362348 M, meaning the concentration of substrate at Vmax is 0.0476724696 M. Though the concentrations of stock solutions of substrate may exceed this concentration, in solution the concentration of total substrate is less than this amount, meaning the dynamic range of lactose concentration, in addition to the constant ONPG concentration, does not exceed the concentration of substrate at Vmax and that our assay is a valid means of detecting lactose in this range. Equation 1. Lineweaver-Burk equation, which is the adjusted Michaelis-Menten equation in the form y=mx+b so that it can easily be plotted and analyzed. Figure 7: The Lineweaver Burke plot, or adjusted Michaelis Menten equation (Equation 1) was plotted above. Then, the y-intercept and slope were determined in order to calculate Km and the concentration of substrate at Vmax for lactase. Note these values may differ from other known values because our lactase is merely crushed Lactaid pills. Conclusion: The purpose of this research was to determine if it is possible to develop a qualitative assay to assess whether a sample containing an unknown concentration of lactose is below or above the threshold of an individual’s tolerance. Our research would then be to determine if it is valid to create an assay in which quantifying the effects of varying concentrations of lactose on ONPG due to competitive inhibition may be compared in order to qualitatively classify lactose concentrations. This goal was ultimately achieved and supported. Rephrase, not really a hypothesis. In ONPG range finding, the 0.033 M ONPG solution was found to be the optimal concentration of ONPG for future testing because it resulted in a strong yellow color in the 2-3 minute range. Moreover, it was very close to the cutoff concentration of lactose (1% or 0.029M), so determining the rate of reaction of lactase with this concentration would be very similar to that of lactose at this concentration and would be easier to detect. Then, lactase range finding led to the discover that the 1:100 concentration of stock solution, or approximately 1:1000 concentration of lactase to buffer, would be optimal because it allowed for a steady, constant rate of reaction of ONP produced over the optimal time range for the assay. The first experiment in assay development revealed the optimal concentration of ONPG in the assay would be 0.0018125 M, as in this case ONPG acts as an indicator (juxtaposed to the previous experiment where ONPG also replicated lactose). This is because the reaction resulted in a fairly constant rate of reaction for the entirety of the optimal time range, and produced a maximum absorbance in the middle of the range of detection for the spectrometer. In experiment two, as the concentration of lactose increased, the rate of reaction of lactase, as determined by rate of ONP produced, decreased. This is because lactose acts as a competitive inhibitor with ONPG, as they both “compete” for the active site of the enzyme. As a result, with more lactose, there is more inhibition, and therefore a faster rate of reaction. Finally, in experiment 3, as lactase was constant and ONPG varied. This allowed for the determination of Km and the concentration of substrate at Vmax to be 0.0238362348 M and 0.0476724696 M respectively, therefore verifying that the dynamic range of lactose, in addition to the constant concentration of ONPG, does not exceed the concentration of substrate at Vmax. Finally, the results from this research effectively confirm the initial hypothesis. Because lactose acts as a competitive inhibitor with ONPG for the lactase enzyme, the concentration of lactose can be directly correlated with the rate of reaction of ONP produced. Moreover, the dynamic range for the final assay was confirmed to be valid as well–0.125% to 8%, which reflects the range of lactose to be potentially detected in foods, does not exceed the concentration of substrate at Vmax, therefore confirming that the initial rate of reaction of the enzyme continues to vary and therefore correlate to lactose concentration in this range. Future Research: Future research involves both homogenization of foods and product development. Homogenization of foods, whether mechanical or chemical, is necessary in order to put foods into solution so that they can be used in the assay without disrupting the enzymatic process and spectrophotometry. Moreover, the goal of the final product is for it to be small, portable, and user-friendly. Our current ideas for a final product are modeled after the very compact Nima Glucose Sensor, as pictured to the right (Nima 2017).

References

1. Borralho, Tracy, et al. “Lactose Induction of the Lac Operon in Escherichia Coli B23 and Its Effect on the o-Nitrophenyl ß-Galactoside Assay.” Journal of Experimental Microbiology and Immunology (JEMI) , vol. 2, Apr. 2002, pp. 117–123., www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/2/2-117.pdf.
2. Carper, Steve. “The Really BIG List of Lactose Percentages.” Lactose Percentages of Dairy Products, 2017, www.stevecarper.com/li/list_of_lactose_percentages.htm.
3. “Lactose Intolerance – Genetics Home Reference.” U.S. National Library of Medicine. National Institutes of Health, n.d. Web. 27 Apr. 2017. <https://ghr.nlm.nih.gov/condition/lactose-intolerance>.
4. “Nima Starter Kit.” Nima, the Portable Gluten Sensor. N.p., n.d. Web. 27 Apr. 2017. <https://shop.nimasensor.com/products/nima-starter-kit&gt;.
5. 2/20/13, Chm333 Lectures 15:, Spring 2013, and Professor Christine Hrycyna. Of an Enzyme Catalyzed Reaction Is Dependent upon the Substrate Concentration [S]. Velocity Related to [S] (n.d.): n. pag. Web.