During the experiment substrate with high density, collected at 70 min, led to increased calcification of their matrix.
In the Na+ uptake experiment, substrate ions bind less well from the outside at low external pH due to proton competition.
The choice of substrate depends on the goals of the experiment: substrate I or III when wide variations in substrate concentrations are needed but high sensitivity is not; substrate IV when high sensitivity is needed.
Deuterium incorporation experiment: Substrate IF (100 μ m) was incubated with enzyme (89 μg mL−1) in phosphate buffer (50 m m) in H2O containing EDTA (5 m m) at 37 °C at pD 6.5 in H2O.
In all experiments, substrate-target gap was set at 10 cm, magnetron target current was 0.1 A, base pressure was 5 ⋅ 10− 4 Pa and work pressure was (4 ± 1) 10−1 Pa.
Gluc luminescence is linear related to substrate (coelenterazine) concentration between 0.1 and 10 µM (Wille et al. 2012); for this reason, in all experiments, substrate was added in excess to guarantee that all the enzyme was catalyzing the reaction to produce light for all the experiments.
In some experiments, substrate-free perfusion fluid was used, and trace amounts of [1-C]octanoate (0.01 μCi/mL) were infused from the beginning of the perfusion experiments.
For the first set of experiments, substrate was incubated with buffer or nucleic acid free WT or S489D coilin proteins, followed by DNase treatment.
In the second adhesion experiment, pericardium substrates were equilibrated at various pH levels prior to testing while keeping the adhesive precursors at pH 7.4.
In the second adhesion experiment, pericardium substrates were equilibrated at various pH levels prior to testing while keeping the adhesive precursor solution buffered at pH 7.4.
