Assay Development for Metal-Dependent Enzymes-Influence of Reaction Buffers on Activities and Kinetic Characteristics

Abstract

Buffers are often thought of as innocuous components of a reaction, with the sole task of maintaining the pH of a system. However, studies had shown that this is not always the case. Common buffers used in biochemical research, such as Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), can chelate metal ions and may thus affect the activity of metalloenzymes, which are enzymes that require metal ions for enhanced catalysis. To determine whether enzyme activity is influenced by buffer identity, the activity of three enzymes (BLC23O, Ro1,2-CTD, and trypsin) was comparatively characterized in N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), Tris-HCl, and sodium phosphate buffer. The pH and temperature optima of BLC23O, a Mn²⁺-dependent dioxygenase, were first identified, and then the metal ion dissociation constant (K_d) was determined in the three buffer systems. It was observed that BLC23O exhibited different K_d values depending on the buffer, with the lowest (1.49 ± 0.05 μM) recorded in HEPES under the optimal set of conditions (pH 7.6 and 32.5 °C). Likewise, the kinetic parameters obtained varied depending on the buffer, with HEPES (pH 7.6) yielding overall the greatest catalytic efficiency and turnover number (k_cat = 0.45 ± 0.01 s^-1; k_cat/K_m = 0.84 ± 0.02 mM^-1 s^-1). To corroborate findings, the characterization of Fe³⁺-dependent Ro1,2-CTD was performed, resulting in different kinetic constants depending on the buffer (K_{m (HEPES, Tris-HCl, and Na-phosphate)} = 1.80, 6.93, and 3.64 μM; k_{cat(HEPES, Tris-HCl, and Na-phosphate)} = 0.64, 1.14, and 1.01 s^-1; k_cat/K_{m(HEPES, Tris-HCl, and Na-phosphate)}= 0.36, 0.17, and 0.28 μM^-1 s^-1). In order to determine whether buffer identity influenced the enzymatic activity of nonmetalloenzymes alike, the characterization of trypsin was also carried out. Contrary to the previous results, trypsin yielded comparable kinetic parameters independent of the buffer (K_{m (HEPES, Tris-HCl, and Na-Phosphate)} = 3.14, 3.07, and 2.91 mM; k_{cat(HEPES, Tris-HCl, and Na-phosphate)} = 1.51, 1.47, and 1.53 s^-1; kcat/K_{m (HEPES, Tris-HCl, and Na-phosphate)} = 0.48, 0.48, and 0.52 mM^-1 s^-1). These results showed that the activity of tested metalloenzymes was impacted by different buffers. While selected buffers did not influence the tested nonmetalloenzyme activity, other research had shown impacts of buffers on other enzyme activities. As a result, we suggest that buffer selection be optimized for any new enzymes such that the results from one lab to another can be accurately compared.

Figure 1

Reactions catalyzed by intradiol (A) and extradiol (B) dioxygenases. Image was generated using ChemDraw.

Figure 2

pH optima of BLC23O in HEPES (A), Tris-HCl (B), and in phosphate (C) buffers. Reactions contained 50 mM of the respective buffers, 1 mM 3-methylcatechol, 10 μM Mn²⁺ (except for reactions in phosphate that contained 100 μM Mn²⁺), and 18.6 μg/mL BLC23O. Absorbance was monitored at 388 nm for 60 min at 32.5 °C and read every 30 s. Reactions were performed in triplicate, and the absorbance was corrected against the blank. The initial linear slope was used to calculate the specific activity using the Beer–Lambert equation. Error bars represent the standard deviation.

Figure 3

BLC23O temperature profile in HEPES (A), Tris-HCl (B), and phosphate (C) buffer. Reactions in HEPES were performed at pH 7.6, in Tris-HCl at pH 7.4, and in phosphate at pH 7.2. Each reaction contained 50 mM buffer, 1 mM 3-methylcatechol, 10 μM Mn²⁺ (100 μM Mn²⁺ in phosphate buffer), and 18.7 μg/mL BLC23O enzyme. Kinetic curves were collected at 388 nm for 60 min and read for every 30 s interval. The corrected absorbance against the blank was used to determine the slopes to calculate the specific activity under each condition.

Figure 4

Dependence of BLC23O activity at varying concentrations of Mn²⁺ in the optimal conditions for HEPES (blue), Tris-HCl (orange), and Na-phosphate (gray). The 250 μL reactions (n = 3) contained 50 mM of the respective buffers, 1 mM 3-methylcatechol, 18.7 μg/mL BLC23O, and varying concentrations of MnCl₂·4H₂O. Reactions were initiated by the addition of 3-methylcatechol and were monitored at λ = 388 nm for 60 min. The specific activity was calculated by using the blank-corrected absorbance. The mean specific activity ± standard deviation is shown, and the dashed lines represent the specific activity curve model created with the Solver add-in from Microsoft Excel. An axis break is shown between 60 and 180 μM Mn²⁺. The graph was created by using OriginLab.

Figure 5

Kinetic curves of BLC23O in HEPES (orange), Tris-HCl (blue), and Na-phosphate (gray) under the same experimental conditions (pH 7.4 and 32.5 °C). The 250 μL reactions (n = 3) contained 50 mM of the respective buffers, varying concentrations of 3-methylcatechol, 18.7 μg/mL BLC23O, and MnCl₂·4H₂O (10 μM in HEPES and Tris-HCl and 100 μM in Na-phosphate). Reactions were initiated by the addition of 3-methylcatechol and were monitored at λ = 388 nm for 60 min. The specific activity was calculated using the blank-corrected absorbance. The mean specific activity ± standard deviation is shown, and the dashed lines represent the specific activity curve modeled using the Michaelis–Menten formula and the Solver add-in from Microsoft Excel. The graph was generated by using OriginLab.

Figure 6

Catalytic efficiency of BLC23O in relation to K_d (enzyme affinity for metal ion cofactor: Mn²⁺) (A) and in relation to K_m (enzyme affinity for the substrate: 3-methylcatechol) (B) for each experimental condition.