Characterization of Serine Protease Inhibitor from Solanum tuberosum Conjugated to Soluble Dextran and Particle Carriers.

A serine protease inhibitor was extracted from potato tubers. The inhibitor was conjugated to soluble, prefractionated dextran and titanium dioxide and zinc oxide nanoparticles. Conjugation to dextran was achieved by periodate oxidation of the dextran, followed by Schiff base coupling to inhibitor amino groups, and finally reduction, whereas the conjugation to the oxide particles was carried out by aminosilanization and carbonyldiimidazole activation. The inhibitory effect of the conjugated inhibitor was compared to that of free inhibitor in solution and with gelatin gel as a direct substrate. A certain degree of inhibitory activity was retained for both the dextran-conjugated and particle-conjugated inhibitors. In particular, the apparent K i value of the dextran-conjugated inhibitor was found to be in the same range as that for free inhibitor. The dextran conjugate retained a higher activity than the free inhibitor after 1 month of storage at room temperature.

Introduction

Endogenous proteases, such as the serine proteases trypsin and chymotrypsin, are essential for the digestion in the host. However, these enzymes can also cause medical problems such as skin inflammation or acute pancreatitis.[1,2] As a general protection, several endogenous protease inhibitors are also expressed, such as the pancreas trypsin inhibitor and α-1-antitrypsin. Protease inhibitors are also present in seeds and tubers in various plant families, including Solanaceae, potato family. As an example, protease inhibitors extracted from Solanum tuberosum, common potato, have inhibiting effects on digestive enzymes such as trypsin, chymotrypsin, and elastase.[3−6] Inhibitory effects on cell growth have also been observed.[7,8] Protease inhibitors have already been successfully tested clinically both in vitro and in vivo.[9]

The wide range of serine protease inhibitors that are expressed in potato (S. tuberosum) and mentioned above serve as a crucial defense mechanism against protein digestion by self, fungal, and bacterial proteases.[10,11] Among these inhibitors, a Kunitz-type inhibitor (potato serine protease inhibitor, PSPI) is the most abundant[11] and accounts for about 20% of the soluble proteins.[12] The configuration of the reactive loop for PSPI is not fully known, but some authors suggest that it represents a two-headed reactive group type; it has also been shown that PSPI can not only bind two different proteases at the same time, i.e., trypsin and chymotrypsin, but also two trypsins due to its two independent binding loops.[10,13,14] The inhibition effect of potato serine protease inhibitor on endogenous proteases has been proven as a remedy against severe dermatitis caused under certain conditions.[15]

A conjugation to selected carriers can increase the convenience of use in different applications,[16−18] e.g., by inclusion in skin protection formulas. Furthermore, conjugation can often have favorable effects on the resistance to high temperatures and harsh chemical environments and may improve the stability of the protein. This study describes the conjugation of PSPI to dextran and to solid carriers, i.e., zinc oxide and titanium dioxide particles, all of which are used in medical and hygiene applications. To the best of our knowledge, no immobilization protocol for PSPI has been reported so far. Dextran is biocompatible[19] and has previously been documented as an excellent carrier for different proteins and is here chosen for this purpose.[20,21,24,25] Among the different conjugation methods between dextran and proteins,[24−31] the periodate oxidation method is effective, feasible, and thus well-established.[20] Furthermore, it does not lead to a direct dextran–dextran cross-linking. In this study, prefractionated water-soluble dextran with 150–190 kDa molar mass was used to conjugate PSPI by the periodate oxidation method, and the result of the conjugation was analyzed by a combination of light-scattering, chromatography, and concentration measurements.[21] The conjugation to the oxide particles was carried out by aminosilanization followed by carbonyldiimidazole (CDI) activation.[22,23]

Our aims in the present work were to (1) establish conjugation protocols for the protease inhibitor from S. tuberosum onto soluble and particle carriers and (2) characterize the stability and kinetics parameters of the conjugated PSPI.

Results and Discussion

Dextran Size Refractionation, Quantification, and Light-Scattering Analysis

The dextran concentration and mass distribution in the eluates of size exclusion chromatography are shown in Figure a, and the dextran concentration in the major fractions was between 6 and 12.8 mg/mL. The sensitivity limit of the sugar measurement by the 3,5-dinitrosalicylic acid (DNS) method is 0.3 mg/mL dextran based on a standard curve. The light-scattering analysis of unfractionated dextran T70 (Figure b) displays a major mass spread from 151 to 60 kDa. The fractionation by size exclusion chromatography resulted, as expected, in a narrower size range. Oxidized dextran displayed a generally later elution peak compared to the intact dextran (Figure c).

Figure 1

(a) Dextran concentration measured by the DNS assay (●) and molecular weight determined by light scattering (□) as a function of elution volume (mL). (b) Light-scattering chromatogram of dextran 10 mg/mL and (c) light-scattering chromatogram of oxidized dextran 10 mg/mL. The shift to the right in elution position for the oxidized dextran is demonstrated by the vertical black line. Upper (red) line represents scattering intensity, flat (green) line represents absorbance at 280 nm, and the lowest (blue) line represents differential refractive index.

The analyzed peak of each sample is divided into front, peak, and back to highlight the distribution of the molecular weight of each sample in the peak. The value of each sample peak as a whole is also included. The oxidation of dextran resulted in a decrease in molecular weight from 68 to 17 kDa, indicating that a degradation has taken place (Table ), assuming the same value for the specific refractive index increment dn/dc. An example of one conjugate (alt 1) is also shown in Table to illustrate the larger molecular weight of the conjugate. The theoretical mass averages for the total sample contents were calculated using the well-known expressions below (eq ).

Table 1

Light-Scattering Analysis of Dextran and Its Derivatives (kDa)

	Mw front	Mw peak	Mw back	Mw whole
dextran, 10 mg/mL	116	61	31	68
oxidized dextran, 10 mg/mL	32	11		17
alternative 1, conjugate 40 μM	658	182		396

Conjugation of Oxidized Dextran and PSPI

The conjugation was carried out using five different alternatives following the same general principle. The alternatives differed in the ratio between oxidized dextran and PSPI, time of incubation, amount of reducing agent, and whether the alternatives were neutralized with HCl. All data regarding the conjugation can be found in Table . In the analysis of the conjugation according to alternative 1, the size-exclusion chromatography showed two elution peaks (Figure a), one for conjugate and one for nonconjugated inhibitor, probably due to the considerably high PSPI/oxidized dextran molecule ratio (6.95) and the initial presence of 0.1 M NaBH3CN. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that this conjugate stayed close to the top of the separation gel, confirming a very large size (Figure a, lane 2) compared to that of the free PSPI (Figure a, lane 3). Light-scattering analysis (Figure b and Table ) revealed an average molecular weight of the same conjugate of approximately 400 kDa. It is obvious, both from scattering data and chromatogram itself, that the conjugation resulted in a considerable increase in molecule size and mass. This prominent increase in molecule size and mass can not be explained by the formation of isolated complexes between proteins and single dextran molecules but may reveal a degree of protein-mediated cross-linking. As can be seen in Figures ,5, and 6, there are 10 lysine residues (marked in magenta in the structure) available for conjugation but thus also for cross-linking. The formation of the bond between oxidized dextran and PSPI is shown in detail in Figure .

Table 2

Conjugation Condition for Oxidized Dextran and PSPI with Each Conjugate Having the Corresponding Apparent Ki Value

alternative	PSPI dextran mass ratio	incubation (h)	amount NaBH3CN	NaBH4	HCl to neutralize	Ki (μM)	efficiency
free PSPI						0.102 ± 0.025	1.00a
1	0.59:1	2	100 mM, present throughout	no (stopped by Tris, pH 9.0)		0.277 ± 0.032	0.37
2	0.18:1	22	1 mM, last 2 h	100 mM, 30 min	yes	0.233 ± 0.034	0.44
3	0.18:1	23	no	100 mM, 30 min	yes	0.191 ± 0.025	0.53
4	0.09:1	23	no	100 mM, 30 min	yes	0.171 ± 0.036	0.60
5	0.33:1	23	no	100 mM, 30 min	yes	0.252 ± 0.026	0.41

Reference value.

Figure 3

(a) Size-exclusion chromatogram of conjugation alternative 1 to the left and SDS-PAGE analysis of conjugate alternative 1 to the right. Lane 1: molecular standard ranging between 260 and 3.5 kD; lane 2: the 1st peak from size-exclusion chromatography representing conjugate; lane 3: the 2nd peak from size-exclusion chromatography representing free PSPI. (b) Light-scattering chromatogram of conjugate alternative 1. The marked area was the conjugate peak. Upper (red) line represents scattering intensity, flat (green) line represents absorbance at 280 nm, and the lowest (blue) line represents differential refractive index.

Figure 2

Schiff base formation followed by reduction between oxidized dextran and a primary amine of one lysine on PSPI gives the modeled dextran–PSPI conjugation (not correct scale). PDB ID: 3TC2.

Figure 5

Sequence of PSPI where suggested active sites are marked in pink (I: Asn45-Asp50, II: Ser71-Phe80, III: around Lys95) and the ten possible conjugation sites are marked in magenta. Note that the proposed active site Lys95 is also a possible conjugation site.

Figure 6

Model of PSPI where two of the possible reactive sites (Phe75 and Lys95) of this double-headed inhibitor are marked in pink. The possible conjugation sites (lysine) are marked in magenta. The model is also shown after a 180° flip to better demonstrate possible conjugations sites. PDB ID: 3TC2.

In the conjugation alternative 2, the molar ratio between PSPI and oxidized dextran was 1.67. The size-exclusion chromatography showed that only one peak, eluting generally somewhat later than the first peak observed from alternative 1, but no separated free PSPI peak was observed in the elution volume range expected for that (Figure ). The SDS-PAGE analysis further confirmed this, since the peak pools of the alternative 2 conjugate showed again a major material spread from close to the top of the separation gel with a smear further into the gel, but all of it migrating slower than the free PSPI (Figure , lanes 1–4). These results indicated that all PSPI molecules were conjugated to the oxidized dextran. In conclusion, the procedure in alternative 2 resulted in complete conjugation, possibly due to the long overnight incubation. The conjugation alternatives 3–5 did also result in complete conjugation and a similar inhibitory efficiency (Table ). However, the data reveal, as expected, a certain degree of protein-mediated cross-linking of the dextran carriers.

Figure 4

Size-exclusion chromatography on sephacryl S-300 for dextran–PSPI conjugation alternative 2 to the left. SDS-PAGE analysis for 2nd–5th conjugations to the right. Lanes 1–4: pools 2–5 of the fractionated alternative 2, containing PSPI 6–14 μg; lane 5: molecular standard (260–3.5 kD); lane 6: free PSPI, 16 μg; and lanes 7–9: dextran–PSPI conjugates from alternative 3, 4, and 5, respectively, containing 6–11 μg PSPI.

Inhibition Activity Analysis of Free and Dextran-Conjugated PSPI

The double-reciprocal plots were analyzed by linear regression using GraphPad Prism 8. Ki was calculated according to eqs –3, and the values are listed in Table . Generally, the apparent Ki of the dextran-conjugated inhibitor molecules is approximately twice as high as that for the free inhibitor.

The apparent Ki values of the conjugates are obviously affected of which lysine(s) that serve in the conjugation and how close the conjugation is to the active site(s). It was earlier proposed[13,32] that this double-headed inhibitor could simultaneously bind two of the same or different proteases at the same time. The binding of two trypsins simultaneously to PSPI is definitely confirmed, whereas only one α-chymotrypsin was bound.[14] The active site(s) of PSPI have not been unequivocally identified due to the lack of complex structural data, but there are a number of theoretically proposed active sites (Figure ) that are most commonly found in the protruding loops.

For monomeric Kunitz type serine protease inhibitor, it is most common that the section Ser71-Phe80 serves as an active site loop, with Phe75 being the P1 residue, so there is one proposed active site in PSPI located around Phe75 and, for the same reason, one around Lys95. It can be seen in Figures and 6 that most of the possible conjugation sites most likely do not affect the inhibition of the protease(s). Since Lys95 also is a possible site for conjugation, the possible outcome that a number of the inhibitor molecules are conjugated by means of this residue will obviously affect the Ki value since that site is no longer available on all molecules.

The apparent Ki values are listed below in Table , and the apparent Ki values observed after 1 month of storage at different temperatures can be seen in Table . The apparent Ki values for dextran–PSPI conjugates lie within the same range (μM) and, compared to that of the free inhibitor, the Ki values increased by a factor of 1.7–2.6, which as mentioned above could be due to cross-linking and sterical hindrance. To evaluate the conjugates, we have chosen to use the parameter efficiency as defined below. The efficiency is easily regarded as the fraction of inhibitor molecules that are functionally active, and the loss of efficiency can be ascribed to denaturation and/or sterical inaccessibility. The efficiency is thus calculated from the apparent Ki value observed, . After the conjugation of PSPI to dextran, the inhibitor still remains 39–58% active in the different conjugation alternatives and remains 12–20% active after 1 month of storage. The SDS-PAGE analysis (supplement) of the stored conjugates shows that they still remain intact and there are no traces of released PSPI, irrespective of the temperature of storage. A control experiment of free PSPI stored at RT for 1 month gave an apparent Ki value of 0.902 μM (Supporting Information Figure S6), which shows an efficiency of 0.11 relative the freshly made free inhibitor with an apparent Ki of 0.102 μM (Table ). This shows that the conjugated PSPI are roughly twice as stable at room temperature than the free inhibitor.

Table 3

Apparent Ki Values Observed after 1 month of Storage of the Dextran–PSPI Conjugate Alternative 2 under Controlled Conditions Using Freshly Prepared Free Inhibitor as a Reference

alternative	Ki (μM)	efficiency
fresh free inhibitor	0.102 ± 0.025	1.00a
conjugate stored at RT	0.512 ± 0.112	0.20
conjugate stored at 4 °C	0.889 ± 0.072	0.12
conjugate stored at –20 °C	0.599 ± 0.057	0.17
free inhibitor stored at RT	0.902 ± 0.194	0.11

Reference value.

Inhibition Activity Analysis of PSPI Conjugated to Activated Particles

The amount PSPI conjugated to activated particles was calculated to be 2.86 nmol/mg for TiO2 and 1.70 nmol/mg for ZnO. During the conjugation, the conjugate of the particles and PSPI was exposed to 65 °C for 5 h during the buffer removal stage, and the conjugate still remains active with an apparent Ki value of 1.8 μM for the TiO2–PSPI conjugate and 4.8 μM for the ZnO-PSPI conjugate (Table ). A control experiment where the free PSPI was exposed to the same environment for the same time gave an apparent Ki value of 17 μM, which clearly indicates that the conjugates are more resistant to heat treatment. The efficiency (Table ) of the conjugated inhibitor was thus calculated to be 0.02 for ZnO-PSPI and 0.06 for TiO2-PSPI, which, however, is better than that observed for free inhibitor subjected to the same heat treatment.

Table 4

Apparent Ki Values of Different Conjugates

state of PSPI	Ki (μM)	efficiency
free PSPI	0.10 ± 0.03	1.00a
TiO2-PSPI	1.81 ± 0.27	0.06
ZnO-PSPI	4.83 ± 0.99	0.02
free PSPI after heat treatment	17.3 ± 1.13	0.006

Reference value.

Inhibition Activity Analysis by the Gelatin Erosion Method

The gelatin erosion method was chosen as an additional method to evaluate the function of the conjugate since it mimics a process on a surface that needs a protective cover layer. The free PSPI and −20 °C stored dextran–PSPI conjugate showed inhibition of both trypsin and chymotrypsin digestion actions on gelatin in a 24-well plate. The ratio (vi/v0) between the area increase rate in the presence of inhibitor (vi) and the area increase rate for the undisturbed enzyme (v0) was studied as a function of free PSPI and dextran-conjugated PSPI. The ratio v0/vi (Figures and 8) was a linear function of inhibitor concentration, which is in accordance with competitive inhibition. The linear regression values extracted from the v0/vi graphs revealed that the gelatin erosion method displays a very similar behavior of the free and dextran-conjugated PSPI, which can be seen in Table . The results from the gelatin erosion method show that there is no large difference between the conjugate and free PSPI. This indicates that the conjugation itself of PSPI does not affect the mode of action and that the conjugated inhibitor still can function similarly to the free PSPI in this type of experiment. The mechanism itself (competitive inhibition) is retained, since the v0/vi of the conjugate remains as a linear function of inhibitor concentration.

Figure 7

Inhibition of trypsin by free PSPI (a) and conjugated PSPI (b) studied by the gelatin erosion method.

Figure 8

Inhibition of chymotrypsin by free (a) and conjugated PSPI (b) studied by the gelatin erosion method.

Table 5

Linear Regression Values Extracted from the v0/vi Graphsa

enzyme	state of PSPI	slope from v0/vi
trypsin	free	2.77 ± 0.35
	conjugated	2.54 ± 0.17
chymotrypsin	free	0.76 ± 0.07
	conjugated	0.51 ± 0.05

Values Extracted from GraphPad Prism 8.

Conclusions

The SDS-PAGE analysis of the conjugates showed no traces of a band corresponding to the free inhibitor, proving that the inhibitory effects observed really depended on the conjugated inhibitor. The apparent Ki was influenced by the conjugation, most likely by the sterical hindrance, but increased at most by a factor of 2.6 for the fresh dextran conjugate. Conclusively, half of the inhibitor molecules are, on the average, still accessible. There are 10 lysine residues, i.e., possible conjugation sites, on PSPI where one lysine also is postulated as a possible active site. The Ki value is affected by this random linkage between PSPI and activated dextran. Overall, the conjugation of PSPI leads to an increase in the apparent Ki value but still in an acceptable range. The conjugates showed an improved storage stability compared to the free inhibitor. Similarly, it was found that the conjugation of the inhibitor to the particles makes it more resistant to harsh heat treatment. The different conjugates will thus improve the applicability of the inhibitor both for medical/care purposes and use in biotechnology processes. The change in apparent Ki does, most certainly, reflect the limited availability of individual inhibitor molecules rather than a gradual change of the intrinsic properties of the molecules regarding both kinetics and binding equilibrium.

Materials and Methods

Chemicals

Potatoes and commercial household gelatin powder (type A, from porcine source, Bloom number 220–240 g), of the brand Törsleff’s “favorit gelatin pulver—extra guld”, were purchased from the local food shop. 3,5-Dinitrosalicylic acid (DNS), K-Na tartrate, Dextran T70, sodium periodate (NaIO4), sodium cyanoborohydride (NaBH3CN), sodium borohydride (NaBH4), polyacrylamide, Coomassie Brilliant Blue R-250, Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPA), titanium dioxide, zinc oxide, aminopropyltriethoxysilane, carbonyldiimidazole, trypsin, and chymotrypsin were all purchased from Sigma-Aldrich. Protein molecular weight standard kit (260–3.5 kD) was purchased from Norvex. All other chemicals were of analytical grade.

Preparation of PSPI

The crude extraction and chromatographic separation were principally carried out according to the study by Pouvreau and Valueva.[11,33] Potatoes were mixed with MQ water, followed by the precipitation of ammonium sulfate. After heat-shocking of the resuspension at 56 °C for 15 min, the supernatant was reprecipitated by ammonium sulfate, and the resuspension was applied to desalting, cation exchange, and size-exclusion chromatography. The major fractions with high inhibition activity of PSPI in 0.1 M phosphate-buffered saline (PBS), pH 7.4, were collected and stored at −20 °C after the absorbance measurement at 280 nm. A value of 27305 M–1 cm–1 for the molar extinction coefficients was used for the calculation of protein concentration.

Preparation of Dextran

Dextran Quantitation by the DNS Method

DNS reagent was prepared from 3,5-dinitrosalicylic acid (DNS), K-Na tartrate, and NaOH.[34] Dextran T70 water solutions with concentrations ranging from 0.3 to 30 mg/mL were used as standards and MQ water as a control. Dextran samples (0.1 mL) were prehydrolyzed by boiling for 1 h with 1 mL of 1 M HCl and neutralized with 1 mL of 1 M NaOH after cooling. Then, 0.5 mL of the sample was mixed with 0.5 mL of DNS reagent. After boiling for 5 min and cooling, 4.5 mL of deionized water was added to the mixture and the absorbance at 540 nm was measured.

Dextran Prefractionation

A volume of 38 mL dextran T70 with concentrations of 30 mg/mL in MQ water was fractionated by size-exclusion chromatography on a sephacryl S-300 (2.6 × 60 cm2, GE Health Care, Uppsala, Sweden) column at a flow rate of 0.2 mL/min (Figure a). The dextran concentration in the fractions was determined by the DNS method.[34] Two consecutive 7 mL fractions in the dextran peak were pooled and stored at −20 °C.

Dextran Mass Determination

Light-scattering analysis was performed by injecting 200 μL of dextran or dextran derivatives solutions via a 200 μL of loop into a Superdex 200 column (10 × 300 mM) connected sequentially with an interferometric refractometer (Wyatt technology, Optilab DSP), enhanced optical system (DAWN EOS, Wyatt technology), and UV flow spectrometer to determine the molar mass. A srii (dn/dc) of 0.144 mL/g was used for the molar mass calculation. dn/dc is an easily measured change in n with a change in c (weight concentration, g/mL).

Conjugation of PSPI to Dextran

The prefractionated dextran pool was incubated with 50 mM NaIO4 for 1 h at room temperature, and the oxidized dextran was separated from the salts by gel chromatography on PD-10 columns using MQ water as an eluent. PSPI was then mixed with the oxidized dextran and incubated at selected times with or without the simultaneous presence of sodium cyanoborohydride (NaBH3CN) (Table ).

Alternative 1: 10 mL of oxidized dextran (5.3 mg/mL) from the 190 kD pool was mixed with 10 mL of potato protease inhibitor at 194 μM. The reaction was carried out in the presence of 0.1 M NaBH3CN at room temperature for 2 h and terminated by the addition of one-fifth volume of 1 M Tris–HCl, pH 9.0.

Alternative 2: 10 mL of oxidized dextran (8.7 mg/mL) from the 150 kD pool was mixed with 5 mL of PSPI at 194 μM. The mixture was kept at room temperature overnight (20 h). NaBH3CN was then added at a final concentration of 1 mM and the mixture was kept at room temperature for further 2 h. The reaction was stopped by the addition of sodium borohydride (NaBH4) at the final concentration of 0.1 M for 30 min, followed by neutralization to pH 7.0 by the addition of 1 M HCl. The resulting solution was filtered through a 0.2 μm membrane and subjected to size-exclusion chromatography on Sephacryl S-300 at a flow rate of 0.5 mL/min. The peak fractions were collected in five pools (pool 1: 164–179 mL, pool 2: 180–203 mL, pool 3: 204–243 mL, pool 4: 244–283 mL, pool 5: 284–307 mL). The absorbance at 280 nm of the fractions was measured, and those in the first peak, corresponding to the dextran-conjugated PSPI, were collected. The pools were stored at −20 °C.

Alternatives 3–5: PSPI at 194 μM was mixed with 8.7 mg/mL oxidized dextran at 150 kD with the volume ratios of 0.5:1, 0.25:1, and 0.1:1, respectively. The mixtures were incubated at room temperature for 23 h, followed by the addition of NaBH4 and HCl as above. No NaBH3CN was used.

SDS-PAGE Analysis

PSPI and dextran conjugates were analyzed by SDS-PAGE on 13% polyacrylamide gel. The gel was stained with 0.5 mg/mL Coomassie Brilliant Blue R-250 in a solvent of 10% (v/v) acetic acid, 40% (v/v) methanol, and 50% (v/v) MQ water and destained using the same solvent. Molecular standard (260–3.5 kD) from Norvex was used.

Conjugation of PSPI to Inorganic Particles

Derivatization of Particles

All derivatization and immobilization steps were performed at room temperature in plastic falcon tubes. The inorganic particle carriers (TiO2 and ZnO) (0.5 grams) were first silanized using 100 mM APTES in 10 mL of ACN while stirring for 24 h. The silanized particles were then centrifuged, followed by the removal of ACN, washing three times with EtOH, one final time with acetone, and then drying at 65 °C overnight. The particles were activated using 120 mg of CDI and 0.72 mmol triethylamine in 5 mL of ACN while stirring for 3 h. Then, the particles were centrifuged to remove ACN, washed three times with EtOH, one final time with acetone, and then dried at 65 °C overnight.

Immobilization of PSPI

Immobilization of PSPI was carried out according to Table . The particles were collected by centrifugation, and the absorbance was measured at 280 nm for the supernatants. The remaining nonconjugated protein was determined. The immobilization was terminated by washing the particles with 0.1 M NH4HCO3 pH 7.52 followed by drying at 65 °C for 5 h and storage at 4 °C until further use.

Table 6

Conjugation Conditions for Activated Particles and PSPI

	amount activated particles (mg)	PSPI (nmol)	time of reaction	buffer
TiO2	100	482	24 h, while stirring	0.1 M NH4HCO3 pH 7.52
ZnO	100	448		0.1 M NH4HCO3 pH 7.52

Kinetics

Inhibition Activity Analysis of Free and Dextran-Conjugated PSPI

Kinetic parameters were obtained by measuring the initial velocities in the presence of varying concentrations of BAPA (0–3 mM) mixed with free (0.18–0.34 μM) or dextran-conjugated PSPI (0.18–0.34 μM). All reactions were performed in 0.1 M NH4HCO3 buffer, pH 7.52, and 0–5% (v/v) DMSO. The reaction was monitored at 410 nm in a UV-1601 UV–vis spectrophotometer (Shimadzu). The apparent Ki was extracted after fitting the data to linear regression in GraphPad Prism 8 according to the Lineweaver−Burk equation (eq ) and its extended version (eq ).

End Point Measurements for Inorganic Particle Conjugates

Kinetic parameters were obtained by incubating substrate, enzyme, and conjugate in a plastic Eppendorf tube with an end-over-end rotation. The concentrations of TiO2-PSPI varied between 2.65 and 5.71 μM, ZnO-PSPI between 1.70 and 3.40 μM, and BAPA between 0 and 2 mM. All reactions were performed in 0.1 M NH4HCO3 buffer, pH 7.52, and 0–5% (v/v) DMSO. One hundred fifty microliters of aliquots were withdrawn at 5 time points from each sample, and the reaction was stopped in 100 mM acetic acid, pH 2.8. Absorbance was measured at 410 nm in a UV-1601 UV–vis spectrophotometer (Shimadzu). The apparent kinetic parameter Ki was extracted after fitting the data to linear regression in GraphPad Prism 8.And its extended versionwhere Km,app = Km × (1 + [I]/Ki)

For alternatives 3–5, Ki was calculated according to the following equation

Inhibition Activity Analysis by the Gelatin Erosion Method

The method was principally described earlier.[35] Gelatin powder (type A, from porcine source, Bloom number 220–240 g) with a concentration of 0.04 g/mL was dissolved in 0.1 M PBS, pH 7.4, at 38 °C. After cooling to 30 °C, it was mixed with free or conjugated PSPI, 0.1 M PBS, and MQ water to get a gelatin concentration of 0.02 g/mL and a series of PSPI concentration. A volume of 1 mL of mixture was added to a 24-well plate in quadruplicates for each PSPI concentration. After solidification at 4 °C for 1 h, a volume of 20 μL of 15 μM trypsin or 20 μL of 15 μM chymotrypsin in 0.1 M PBS, pH 7.4, was pipetted on the center of the gelatin layer and the plates were covered with lids and incubated at 4 °C for 24 h. The diameter of the erosion in the well was measured and the specific enlargement of the erosion area was calculated by subtracting the area that was observed when 20 μL of water was added instead of the enzyme solution.

The relative area growth rate (vi/v0) was analyzed as a function of the inhibitor concentration by GraphPad Prism 8 using the one-phase decay algorithm (eq ).where K is the rate constant.