<i>In Situ</i> Observation of Chymotrypsin Catalytic Activity Change Actuated by Nonheating Low-Frequency Magnetic Field

Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out <i>in situ</i> during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16–410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin–GM-MNPs/trypsin inhibitor–GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme–inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions

N-glycosylated peptides and glycan structures found by the GlycoMod tool in ACE from lung and seminal fluid using MS data.

<p>N-glycosylated peptides and glycan structures found by the GlycoMod tool in ACE from lung and seminal fluid using MS data.</p

Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.

<p>^a Acrylamide adduct on cysteine.</p><p>^b Oxidized methionine.</p><p>^c Contains one or two missed cleavage(s) by trypsin.</p><p>Peptides that contain potential N-glycosylation sites are shown in bold.</p><p>Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.</p

The structures of N and C domains of ACE with potential glycosylation sites and epitopes for mAbs.

<p>Human N domain structure was based on PDB P2C6N and C domain structure—based on PDB 1O86. The epitopes were marked on the N and C domains according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref027" target="_blank">27</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref031" target="_blank">31</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref041" target="_blank">41</a>]. The positions of the epitopes for some mAbs (12 out of 17) are shown by circles on both sides of domain globule. The potential sites of N-glycosylation, 9 on the N domain and 6 on the C domain, are marked by green; Asn494 on the N domain is not seen while Asn1196 is not present in structure of the C domain. The glycosylation sites which might be differently glycosylated in seminal fluid ACE and lung ACE are shown by arrows. Some amino acid residues are shown by numbers according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref038" target="_blank">38</a>] for orientation.</p

Effect of different additives on mAbs binding to seminal fluid and lung ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding of tested compounds to purified seminal fluid and lung ACEs with that without additives. <b>(A)</b> Effect of 20% of human heat-inactivated plasma. <b>(B</b>) Effect of 80% 3 kDa filtrate of human citrated plasma. <b>C</b>-<b>D</b>. Effect of bilirubin (150 ug/ml) in the absence (<b>C</b>) or presence (<b>D</b>) of human albumin at 8 mg/ml concentration (which correspond to its concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p

Amino acid sequences of the lung and seminal fluid ACEs.

<p>Peptides identified by MALDI TOF MS are shown in bold; potential sites of trypsin cleavage are underlined; potential glycosylation sites are marked by green; zinc-recognizing motives are marked by red; putative glycopeptides are shaded.</p

Effect of human plasma, seminal fluid and albumins on mAbs binding to ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding heat-inactivated human citrated plasma, heat-inactivated seminal fluid, as well as human and bovine albumins to purified seminal fluid and lung ACEs with that without additives. <b>A-B</b>. Effect of 20% of heat-inactivated human plasma (<b>A</b>) and heat-inactivated seminal fluid (<b>B</b>); <b>C-D</b>. Effect of human (<b>C</b>) and bovine (<b>D</b>) albumins at concentrations of 8 mg/ml (similar to albumin concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p

Primary immune response in mice to pure somatic ACE from seminal fluid.

<p>Culture fluids from 670 post-fusion cell populations grown in 96-well plates (primary screening) were anylyzed for the presence of antibodies to seminal fluid ACE and lung ACE in parallel in plate precipitation assay (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>). Presence of antibodies to seminal fluid and/or lung ACE was detected in 91 wells by precipitated ACE activity and the data are presented as the ratio of ACE activity precipitation from seminal fluid ACE to that from lung ACE (SF/Lung ratio). Discrimination of these two ACEs by antibodies from these positive wells was observed in a wide range (A). Besides expected antibodies, recognizing both ACEs (C) with SF/Lung ratio in the region from 0.5 to 1.5, we identified significant proportions of antibodies which preferentially recognized seminal fluid ACE (B) with SF/Lung ratio more than 1.5, and antibodies which preferentially recognized lung ACE (D) with SF/Lung ratio less than 0.5, correspondingly.</p

Effect of anti-catalytic mAbs on the activity of pure seminal fluid and lung ACEs.

<p>Pure seminal fluid and lung ACEs (5 mU/ml with ZPHL as a substrate) were incubated with mAbs (10 ug/ml), which are anti-catalytic for the N-domain active center, i2H5 and 3A5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref027" target="_blank">27</a>], and for the C domain active center, 1E10 and 4E3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref030" target="_blank">30</a>], of ACE. Residual ACE activity was determined with substrates HHL (<b>A</b>) and ZPHL (<b>B</b>) and is presented as the ratio of ACE activity in the presence of mAbs to that without mAbs. Data are also presented as ZPHL/HHL ratio of ACE activity in the presence of mAbs to that without mAbs (<b>C</b>). Results are the mean ± SD of 2–4 experiments, made in duplicates.</p