Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development.

The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group's reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific ß-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal ß-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.

Novel approach to the measurement of antithyroglobulin antibodies in human serum - application of the quartz crystal microbalance sensors.

Measurement of antithyroglobulin antibodies (TgAb) is an inevitable laboratory tool in the management of thyroid gland diseases. Currently available immunoassays still have limitations underlying the necessity of the introduction of fast, sensitive, and label-free technologies. Our aim was to develop a method for TgAb measurement in human serum based on the quartz crystal microbalance (QCM) technology. We immobilized thyroglobulin on the surface of Attana LNB Carboxyl sensor chip®, prepared standard curve covering the range of 1-50000 kIU/L, and established optimal measurement conditions. The validation included determination of the detection limit (LOD), functional sensitivity, linearity, precision, as well as the comparison with the results of the radioimmunoassay (RIA). The LOD and functional sensitivity were 4.2 kIU/L and 4.7 kIU/L, respectively. The method was linear in the range of 20-10000 kIU/L. The regression equation for comparison with RIA was CQCM= 1.0056 • CRIA- 24.2778, whereby no significant proportional or systematic difference was present. There was a good agreement with RIA in the classification of patients according to the clinical significance of the results. The developed method has advantages over currently available assays in terms of better LOQ, a higher upper limit of linearity, and precision. The characteristics of the developed method unambiguously show that the application of the QCM biosensors offers a highly reliable novel approach for the measurement of TgAb in human serum.

Trypsin activity and freeze-thaw stability in the presence of ions and non-ionic surfactants.

Trypsin is a serine protease with important applications such as protein sequencing and tissue dissociation. Preserving protein structure and its activity during freeze-thawing and prolonging its shelf life is one of the most interesting tasks in biochemistry. In the present study, trypsin cryoprotection was achieved by altering buffer composition. Sodium phosphate buffer at pH 8.0 led to pH shift-induced destabilization of trypsin and formation of a molten globule, followed by significant activity loss (about 70%). Potassium phosphate and ammonium bicarbonate buffers at pH 8.0 were used with up to 90% activity recovery rate after 7 freeze-thaw cycles. The addition of non-ionic surfactants Tween 20 and Tween 80 led to up to 99% activity recovery rate. Amide I region changes, corresponding to specific secondary structures in the Fourier transform infrared (FTIR) spectrum, were modest in the case of Tween 20 and Tween 80. On the other hand, the addition of Triton X-100 led to the destabilization of α-helicoidal segments of trypsin structure after 7 freeze-thaw cycles but also increased protein substrate availability.

Broad range of substrate specificities in papain and fig latex enzymes preparations improve enumeration of Listeria monocytogenes.

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100.