An in vitro reaction was performed to examine the catalytic properties of alkaline phosphatase (ALP) a hydrolase enzyme and the synthetic substrate 4-nitrophenyl phosphate (4-NPP). For every molecule of 4-NPP consumed in the reaction, 1 molecule of 4-nitrophenol (4-NP) and 1 molecule of orthophosphate is produced.
We performed an assay of ALP with 4-NPP as the substrate and the reaction product 4-NP to determine the spectral properties of these reactants, i.e. was there a disappearance of the substrate 4-NPP or the appearance of 4-NP or orthophosphate? If both absorb light which one would be more readily detected, and would these chemicals be likely to interfere with one and other in a spectrophotometric assay?
The rate of an enzyme catalysed reaction is determined by how well the enzymes amino acids "fit" to the substrate; this can be altered by several factors such as pH value, temperature, substrate or enzyme concentrations. The reaction which showed the least absorbance in the assay was used in further tests to determine how various other reactants reacted in alkaline phosphatase with and without enzyme present, at different concentrations, and pH values. Standard initial reaction and initial reaction velocities were calculated to determine how or why the other reactions were catalysed or inhibited in alkaline phosphatase.
Materials and Methods
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Stock solutions of all reagents were prepared by instructors. Lab practical handouts contain lists of reagents and equipment (Rayne, 2010a). Alkaline phosphatase (prepared from bovine intestinal mucosa) was purchased from Sigma (Poole, Dorset, UK) as were all other chemicals.
Absorption spectra of 4-NPP and 4-NP
An Enzyme assay to discover the absorbance spectrum for 4-NPP and 4-NP in ALP was carried out using diluted 4-NPP and 4-NP respectively in steps of 5 or 10 nm in an alkaline buffer of 0.5 M Tris, pH 9.2 into cuvettes with the aim of attaining a wavelength of between 360 nm and 440 nm in the fewest steps possible. A clean cuvette containing water was used to zero the spectrophotometer and set aside to be used before each reading. Buffer and ALP were added to cuvettes with each of the diluted solutions of 4-NPP and 4-NP enzyme respectively being added at the last minute and stirred thoroughly before being put through the spectrophotometer, readings were taken at taken every minute for 5 minutes and results recorded. Once the ideal absorptivity was ascertained the mean of four independent results were recorded and plotted onto a graph.
Alkaline phosphatase catalysed reactions
100 ul Enzyme solution (ALP) was diluted with 900 ul of H20, and 1000 ul 4-NPP and added to cuvettes containing a mixture of various reaction solutions with a maximum of 1.25 mL volume such as 7.2 pH buffer, MgCl2, phosphate, EDTA including a cuvette containing no enzyme. As previously, the enzyme was added last (except in the case of the cuvette containing no enzyme) and stirred thoroughly before being measured for absorptivity using a blank cuvette to zero the spectrophotometer and results recorded over a 25 min period in 5 min increments.
The initial absorbance of the 4-NP increased linearly to a high degree for a short time before dropping off and decreasing, whilst the 4-NPP steadily decreased in absorptivity(Fig 1). In Fig 2 the reaction steadily increased in a linearly way over a 25 min period. In table 1 the MgCl2 reaction steadily increases comparably with the standard reaction whilst subsequent reactions increased slightly but were somewhat retarded in their reactions
Figure 1: Spectral absorbance curves of 4-NPP and 4-NP in alkaline solution.
Figure 2: Time Course of the standard linear reaction of Hydrolysed 4-NPP catalysed by alkaline phosphatase (mean of 3 independent measurements of A400 in increments of 5 min).
Table 1: Initial Reaction Velocities
The spectral absorbance curves of 4-NPP and 4-NP in an alkaline solution at near 400nm are given in Figure 1. In all solutions the enzyme was added last and the spectrophotometer zeroed with a blank before each reading to ensure the initial reaction was recorded accurately. The 4-NP which is the product of 4-NPP produces a very high molar absorptivity Hyperbolic curve, this is because ALP is a hydrolase enzyme which can cleave substrates via chemical reactions involving the addition of water to a chemical bond this is an extremely exergonic reaction , which hydrolyses the substrate and converting it to the product 4-NP and orthophosphate, as the substrate is converted to product the rate of reaction increases until the substrate decreases and the reaction reaches equilibrium and no more product can be made, the reaction then slows and decreases, whereas the Substrate 4-NPP which is lacking a phosphate had saturated the enzyme and reached a steady state relatively quickly meaning 4-NPP had an extremely low molar absorptivity at the same wavelength. It is this low molar absorbance indicated that 4-NPP is not a very good absorber of light and therefore would not interfere with other reactions, which made 4-NPP the best choice to run our alkaline phosphatase catalysed reaction.
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The standard linear reaction in Fig 2 was observed as a constant increase in absorbance over time especially between 2 - 3 min indicating that enzyme had catalysed the substrate, this absorbance steadily started to decrease between 4-5 min indicating the substrate was being exhausted and reaching an equilibrium state
Table 1 shows the initial reaction velocities of the various reactions compared to the standard reaction; it was observed that all of the other reactions except the standard and the MgCl2 had been inhibited in their reactions. EDTA is a metal chelator and will bind divalent (ions or molecules that have a valence of two and can form two covalent bonds with other ions or molecules) such as in MgCl2. Mg2 ions also bind to the zinc in ALP slightly inhibiting the ALP. As the original enzyme preparation already contained both Zinc and Mg2 bound to ALP no more was needed to get full activity, we can then assume that the enzyme became saturated by the metal ions in MgCl , and a lack of free enzyme binding sites left insufficient enzyme to catalyse the other reactions making their activation inhibited.