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; kcat/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 (Km (HEPES, Tris‑HCl, and Na‑Phosphate) = 3.14, 3.07, and 2.91 mM; kcat
(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.