A Convenient, Rapid, Sensitive, and Reliable Spectrophotometric Assay

Home , Adenosine kinase, Adenylate kinase, ADK (gene)

molecules Article AArticle Convenient, Rapid, Sensitive, and Reliable SpectrophotometricA Convenient, Rapid, Assay Sensitive, for Adenylate and Reliable Kinase ActivitySpectrophotometric Assay for Adenylate

KaiKinase Song 1, Yejing Activity Wang 2,*, Yu Li 1, Chaoxiang Ding 1, Rui Cai 2, Gang Tao 1, Ping Zhao 1, Qingyou Xia 1 and Huawei He 1,3,* Kai Song 1, Yejing Wang 2,*, Yu Li 1, Chaoxiang Ding 1, Rui Cai 2, Gang Tao 1, Ping Zhao 1, 1 Qingyou Biological Xia Science1 and Research Huawei Center, He 1,3, Southw* est University, Beibei, Chongqing 400715, China; [email protected] (K.S.); [email protected] (Y.L.); [email protected] (C.D.); 1 [email protected] Science Research (G.T.); Center, [email protected] Southwest University, (P.Z.); Beibei, [email protected] Chongqing 400715, (Q.X.) China; 2 [email protected] Key Laboratory of Silkworm (K.S.); Genome [email protected] Biology, College (Y.L.); of Biotechnology, [email protected] Southwest University, (C.D.); Beibei,[email protected] Chongqing 400715, China; (G.T.); [email protected] [email protected] (P.Z.); [email protected] (Q.X.) 2 3 ChongqingState Key LaboratoryKey Laboratory of Silkworm of Sericultural Genome Scienc Biology,e, Chongqing College of Engineering Biotechnology, and Southwest Technology University, Research Beibei, CenterChongqing for Novel 400715, Silk China;Materials, [email protected] Southwest University, Beibei, Chongqing 400715, China 3 * Correspondence:Chongqing Key [email protected] of Sericultural.cn (Y.W.); Science, [email protected] Chongqing Engineering (H.H.); and Tel.: Technology +86-23-6825-1575 Research (Y.W. Center &for H.H.) Novel Silk Materials, Southwest University, Beibei, Chongqing 400715, China * Correspondence: [email protected] (Y.W.); [email protected] (H.H.); AcademicTel.: +86-23-6825-1575 Editor: Stefano Serra (Y.W. & H.H.) Received: 19 January 2019; Accepted: 12 February 2019; Published: 13 February 2019 Academic Editor: Stefano Serra Abstract:Received: 19Enzymatic January 2019; activity Accepted: assays 12 Februaryare essential 2019; Published:and critical 13 Februaryfor the 2019study of enzyme kinetics. AdenylateAbstract: Enzymatickinase (Adk) activity plays a assays fundamental are essential role in andcellular critical energy for and the nucleotide study of enzymehomeostasis. kinetics. To date,Adenylate assays kinase based (Adk)on different plays aprinciples fundamental have role been in used cellular for energythe determination and nucleotide of Adk homeostasis. activity. Here,To date, we assays show based a spectrophotometric on different principles analysis have beentechnique used for to the determine determination Adk ofactivity Adk activity. with bromothymolHere, we show blue a spectrophotometric as a pH indicator. analysis We analyzed technique the effects to determine of substrates Adk activity and the with pH bromothymol indicator on theblue assay as a pHusing indicator. orthogonal We analyzed design and the effectsthen establ of substratesished the and most the optimal pH indicator assayon for the Adk assay activity. using Subsequently,orthogonal design we evaluated and then establishedthe thermostability the most of optimal Adk and assay the for inhibitory Adk activity. effect Subsequently, of KCl on Adk we activityevaluated with the this thermostability assay. Our results of Adk show and that the inhibitorythis assay effectis simple, of KCl rapid, onAdk and activityprecise. withIt shows this great assay. potentialOur results as show an alternative that this assay to the is simple,conventional rapid, andAdk precise. activity It showsassay. greatOur results potential also as ansuggest alternative that orthogonalto the conventional design is Adk an effective activity assay. approach, Our resultswhich alsois very suggest suitable that for orthogonal the optimization design is of an complex effective enzymeapproach, reaction which conditions. is very suitable for the optimization of complex enzyme reaction conditions.

Keywords:Keywords: enzymaticenzymatic activity activity assay; assay; adenylate adenylate kinase; kinase; spectrophotometry; spectrophotometry; orthogonal orthogonal experiment; experiment; bromothymolbromothymol blue

1.1. Introduction Introduction AdenylateAdenylate kinase kinase (Adk; (Adk; ATP:AMP ATP:AMP phosphotransferase, phosphotransferase, EC EC 2.7.4.3), 2.7.4.3), also also known known as as myokinase, myokinase, isis a a conserved phosphoryl phosphoryl transferase, transferase, which which catalyzes catalyzes the the translocation translocation of of a a phosphoryl phosphoryl group group betweenbetween nucleotides in the reversible reaction (AMP ++ Mg2+•ATP•ATP ↔ MgMg2+2+•ADP•ADP + ADP) ADP) [1]. [1]. Adk Adk is is ubiquitousubiquitous in in different different tissues tissues of of all all living living system systemss and and plays plays a a fundamental fundamental role role in in cellular cellular energy energy andand nucleotide homeostasis. homeostasis. The The hydrogen hydrogen bond bond be betweentween the the adenine adenine moiety moiety and and the the backbone of of Adk is critical for ATP selectivity and can help Ad Adkk recognize the correct substrates in in the complex cellularcellular environmentenvironment [ 2[2].]. Adk Adk is involvedis involved in the in regulation the regulation of cell differentiation,of cell differentiation, maturation, maturation, apoptosis, apoptosis,and oncogenesis. and oncogenesis. Adk mutations Adk inmutations humans causein hu amans severe cause disease a severe called disease reticular called dysgenesis reticular [3]. dysgenesisAdk is regarded [3]. Adk as ais potential regarded target as a potential for medical targ diagnosiset for medical and treatment diagnosis due and to treatment its close correlation due to its closewith othercorrelation diseases, with such other as diseases, aleukocytosis, such as hemolytic aleukocytosis, anemia, hemolytic and primary anemia, ciliary and primary dyskinesia ciliary [4]. dyskinesiaTo date, three [4]. methods To date, have three been methods proposed have to been determine proposed Adk to activity determine based Adk on theactivity detectable based changes on the accompanied with this reaction, such as light absorption, acidity, or the coupled reaction products [5]. MoleculesThe manometric 2019, 24, 1-12; assay, doi: established by Colowick and Kalckar [6], is used www.mdpi.c for the detectionom/journal/molecules of Adk by

Molecules 2019, 24, 663; doi:10.3390/molecules24040663 www.mdpi.com/journal/molecules Molecules 2019, 24, 663 2 of 10

measuring CO2 liberation from a bicarbonate buffer. The reaction catalyzed by Adk is coupled to hexokinase, which speciﬁcally catalyzes the transformation of the terminal phosphate from ADP to glucose. The overall reaction is as follows:

Adk ATP + AMP←−→ADP + ADP (1)

hexokinase ADP + glucose−−−−−−→glucose − 6 − P + AMP + H+ (2)

In the presence of excess hexokinase, the reaction rate is proportional to the Adk concentration. The forward direction reaction is defined as the formation of AMP and ATP from two ADPs, and the reverse direction is defined as the formation of two ADPs from AMP and ATP. Adk activity is conventionally measured in vitro by a spectrophotometric assay. For the forward direction reaction, Chiu et al. [7] have developed a modified assay of Oliver [8] to determine Adk activity by coupling the reaction to hexokinase and glucose-6-phosphate dehydrogenase in which the final product, NADPH, is measured spectrophotometrically at 340 nm. The overall reaction is as follows: H+

Adk ADP + ADP←−→ATP + AMP (3)

hexokinase ATP + glucose−−−−−−→glucose − 6 − P + ADP (4)

glucose − 6 − phosphate dehydrogenase glucose − 6 − P + NADP−−−−−−−−−−−−−−−−−−−−→6 − phosphogluconic acid + NADPH (5)

Adk activity is also measured in the reverse direction by coupling the reaction to pyruvate kinase and lactate dehydrogenase and measuring the oxidation of NADH at 340 nm [5]. The principle of the assay is as follows: Adk ATP + AMP←−→ADP + ADP (6)

Pyruvate kinase Phosphoenolpyruvate + ADP−−−−−−−−−→ATP + Pyruvate (7)

Lactic dehydrogenase Pyruvate + NADH + H + −−−−−−−−−−−−→Lactate + NAD+ (8)

These assays have been used to determine Adk activity for the past decades. However, some disadvantages are also obvious for these assays. Firstly, these assays are time-consuming, multistep processes that require the assistance of other enzymes and are easily subject to errors at each step. Secondly, it is difficult to study the effects of activators and inhibitors on Adk activity with the aid of other enzymes. Finally, the real initial rate of Adk reaction cannot be determined accurately [9]. Therefore, it is necessary to develop a more convenient and accurate assay for Adk activity in vitro. Acid–base indicators are usually applied in enzymatic assay for their extraordinary sensitivity to pH change. In 2002, Yu et al. [10] established an arginine kinase activity assay based on the light absorption of a complex acid–base indicator consisting of thymol blue and cresol red. In the reaction catalyzed by arginine kinase, the produced protons resulted in a decrease in pH of the reaction mixture, thus reducing the absorbance of the mixed indicator in the solution at 575 nm. The arginine kinase activity could be determined according to the change of the absorbance at 575 nm. In the same way, Dhale et al. developed a rapid and sensitive assay to measure L-asparaginase activity with methyl red as an indicator [11]. Bromothymol blue is an excellent indicator as it forms a highly conjugated structure while deprotonated in alkaline solution, resulting in an obvious color change from yellow to blue and the corresponding absorbance change [12]. Enzyme activity is typically influenced by many factors. The traditional method is to do a multifactor analysis that tests all possible combinations of the different factors. However, this takes Molecules 2019, 24, 663 3 of 10 up a lot of time and resources as the number of full factorial experiments is very large. As an Moleculesalternative, 2019, 24 the, x orthogonal design method has been proposed and established. The orthogonal3 of 10 experimental design [13] is a multifactor experiment design assay. It selects representative samples thusfrom arepresenting full factorial assaythe overall in a way situation. that the samplesTherefore, are distributedit is highly uniformly efficient withinfor the the arrangement test range, thus of multifactorrepresenting experiments the overall situation. with optimal Therefore, combination it is highly levels. efficient The for theorthogonal arrangement design of multifactorhas three advantages:experiments (1) with The optimal number combination of tests required levels. to The comp orthogonallete the experiment design has is three relatively advantages: small. (2) (1) The datanumber points of tests are evenly required distributed. to complete (3) the The experiment test results is relativelycan be analyzed small. (2)by Themathematical data points calculation are evenly (e.g.,distributed. range analysis (3) The testand resultsvariance can analysis), be analyzed which by is mathematical particularly calculationuseful to quantify (e.g., range the results. analysis and varianceIn this analysis), study, we which developed is particularly a one-step useful assay to quantify for Adk theactivity. results. It is based on proton generation after Inthe this addition study, of we ATP developed and AMP a one-stepas the substrat assayes, for which Adk activity.can be measured It is based spectrophotometrically on proton generation atafter 614 the nm addition using bromothymol of ATP and AMP blue as as the a substrates,pH indicator. which We caninvestigated be measured four spectrophotometrically factors affecting Adk activity—ATP,at 614 nm using AMP, bromothymol bromothymol blue blue, as a and pH indicator.glycine–NaOH We investigated buffer—at three four levels factors and affecting determined Adk theactivity—ATP, best combination AMP, bromothymol for Adk activity blue, andassay glycine–NaOH by an orthogonal buffer—at experimental three levels design. and determined Finally, thewe evaluatedbest combination the thermostability for Adk activity of Adk assay and by the an inhibitory orthogonal effect experimental of KCl ondesign. Adk activity Finally, with we this evaluated assay. Ourthe thermostability results suggest ofthat Adk this and assay the is inhibitory simple, precis effecte, of less KCl expensive, on Adk activity and a withpotential this assay.alternative Our results to the conventionalsuggest that this enzymes-coupled assay is simple, assay precise, extensively less expensive, used in and clinical a potential and research alternative laboratories. to the conventional enzymes-coupled assay extensively used in clinical and research laboratories. 2. Results 2. Results In this study, Adk activity was determined by a direct and continuous spectrophotometric techniqueIn this without study, coupled Adk activity enzymes. was In determined the enzymati byc a reaction direct and catalyzed continuous by Adk, spectrophotometric the formation of twotechnique ADPs withoutfrom ATP coupled and AMP enzymes. is accompanied In the enzymatic by the reactiongeneration catalyzed of hydrogen by Adk, ions. the Bromothymol formation of bluetwo ADPsis an excellent from ATP acid–base and AMP indicator is accompanied as it form bys the a highly generation conjugated of hydrogen structure ions. while Bromothymol protonated blue in acidis an excellentsolution, acid–baseresulting indicatorin an obvious as it forms color a highlychange conjugated from blue structure to yellow. while The protonated absorbance in acid of bromothymolsolution, resulting blue inat an614 obvious nm is associated color change with from the blue hydrogen to yellow. ion Theconcentration absorbance in of solution. bromothymol Thus, Adkblue atactivity 614 nm can is associatedbe monitored with in the real hydrogen time by ionthe concentration absorbance of in bromothymol solution. Thus, blue Adk at activity 614 nm can in solution,be monitored which in realcan timebe detected by the absorbance by a sensitive of bromothymol spectrophotometer. blue at 614 The nm principle in solution, of whichthis assay can beis illustrateddetected by in a Figure sensitive 1. spectrophotometer. The principle of this assay is illustrated in Figure1.

Figure 1. The principle of the spectrophotometric assay for adenylate kinase (Adk) activity. Figure 1. The principle of the spectrophotometric assay for adenylate kinase (Adk) activity. The effects of substrates and the pH indicator on the assay were analyzed using an orthogonal design,The which effects is of key substrates to establishing and the the pH most indicator optimal on the assay assay for Adkwere activity.analyzed The using factors an orthogonal and levels design,affecting which Adk activityis key to assay establishing are shown the in most Table optimal1. assay for Adk activity. The factors and levels affecting Adk activity assay are shown in Table 1.

Molecules 2019, 24, 663 4 of 10

Table 1. Factors and levels affecting Adk activity assay.

ABCD Level ATP (mM) AMP (mM) Bromothymol Blue (mM) Glycine–NaOH (mM) 1 2.0 1.0 0.0930 0.1 2 2.5 1.5 0.1084 0.3 3 3.0 2.0 0.1238 0.5

2.1. The Maximum Absorption Wavelength of Reaction Mixture A set of nine tests designed by orthogonal experiment is shown in Table2. The absorption spectrum of each set was scanned from 450 to 800 nm in the presence of 5 mM MgAC2. All spectra showed the same maximum absorption located at 614 nm without shift, as shown in Figure2a.

Table 2. Orthogonal array (9, 34) for the analysis of the effects of ATP, AMP, bromothymol blue, and glycine–NaOH buffer on the Adk activity assay.

Factor No. Combination ∆Abs a (0–30 s) ABCD

1 A1B1C1D1 2.0 1.0 0.0930 0.1 0.0914 ± 0.0020 2 A1B2C2D2 2.0 1.5 0.1084 0.3 0.0432 ± 0.0045 3 A1B3C3D3 2.0 2.0 0.1238 0.5 0.0112 ± 0.0017 4 A2B1C2D3 2.5 1.0 0.1084 0.5 0.0404 ± 0.0017 5 A2B2C3D1 2.5 1.5 0.1238 0.1 0.0221 ± 0.0020 6 A2B3C1D2 2.5 2.0 0.0930 0.3 0.0606 ± 0.0022 7 A3B1C3D2 3.0 1.0 0.1238 0.3 0.0341 ± 0.0009 8 A3B2C1D3 3.0 1.5 0.0930 0.5 0.0705 ± 0.0049 9 A3B3C2D1 3.0 2.0 0.1084 0.1 0.0382 ± 0.0017 T1 0.1458 0.1659 0.2225 0.1517 T2 0.1231 0.1358 0.1218 0.1379 T3 0.1428 0.1100 0.0674 0.1221 t1 0.0486 0.0553 0.0742 0.0506 t2 0.0410 0.0453 0.0406 0.0460 t3 0.0476 0.0367 0.0225 0.0407 Range (R) 0.0076 0.0186 0.0517 0.0099 Order C > B > D > A Optimal level A1 B1 C1 D1 Optimal combination A1B1C1D1 a Arithmetic mean of the absorbance changes of bromothymol blue at 614 nm (0–30 s) of three independent tests at each level under the same factor. Ti (T1, T2, T3) is the sum of the recorded absorbance changes (∆Abs) at the same level and under the same factor, and ti (t1, t2, t3) is the arithmetic mean of Ti. The mean of ti represents the inﬂuence of different levels under the same factor on the absorbance of bromothymol blue. Range (R) is the difference between the maximum and the minimum of ti, indicating the effect of each factor on the absorbance of bromothymol blue. The greater the R value, the greater is the inﬂuence of this factor on the absorbance of bromothymol blue at 614 nm.

2.2. Optimization of Adk Activity Assay The effects of ATP, AMP, bromothymol blue, and glycine–NaOH buffer on the Adk activity were analyzed, as shown in Table2. The significance of these factors on the assay was determined by the range (R) value listed in Table2. The results showed that the order of R value was RC > RB > RD > RA. Hence, the significance order of these factors on the assay was C > B > D > A, namely, bromothymol blue > AMP > glycine–NaOH buffer > ATP. Similarly, the significance of different levels on the assay was determined by the ti value listed in Table2. The results showed that the orders were t1 > t3 > t2 for factor A, t1 > t2 > t3 for factor B, t1 > t2 > t3 for factor C, and t1 > t2 > t3 for factor D. Thus, the most optimal combination for Adk activity assay was A1B1C1D1, which was composed of 2 mM ATP, 1 mM AMP, 0.093 mM bromothymol blue, and 0.1 mM glycine–NaOH buffer. Molecules 2019, 24, 663 5 of 10

Next, we conducted a full factorial design for two primary factors of bromothymol blue and AMP at three levels to further optimize the assay. The design matrix and parameters are listed in Table3. The results showed that AB3C1D had the most signiﬁcant absorbance change at 614 nm among all the tested conditions (Table4), indicating AB3C1D (2 mM ATP, 0.6 mM AMP, 0.093 mM bromothymol blue, and 0.1 mM glycine-NaOH buffer) was the most optimal reaction condition for the Adk activity assay.

Table 3. Factors and levels affecting Adk activity assay with the constants of A and D.

ABCD Level ATP (mM) AMP (mM) Bomothymol Blue (mM) Glycine–NaOH (mM) 1 2.0 1.0 0.0930 0.1 2 2.0 0.8 0.0775 0.1 3 2.0 0.6 0.0620 0.1

Table 4. A full factorial design for the analysis of the effects of AMP, bromothymol blue on the Adk activity assay with the constants of A and D.

Factor Run Combination ∆Abs b (0–30 s) ABCD

1 AB1C1D 2.0 1.0 0.0930 0.1 0.0914 ± 0.0020 2 AB1C2D 2.0 1.0 0.0775 0.1 0.0926 ± 0.0030 3 AB1C3D 2.0 1.0 0.0620 0.1 0.0764 ± 0.0030 4 AB2C1D 2.0 0.8 0.0930 0.1 0.0914 ± 0.0020 5 AB2C2D 2.0 0.8 0.0775 0.1 0.0930 ± 0.0018 6 AB2C3D 2.0 0.8 0.0620 0.1 0.0680 ± 0.0041 7 AB3C1D 2.0 0.6 0.0930 0.1 0.0983 ± 0.0028 8 AB3C2D 2.0 0.6 0.0775 0.1 0.0850 ± 0.0054 9 AB3C3D 2.0 0.6 0.0620 0.1 0.0604 ± 0.0001 b Arithmetic mean of the absorbance changes of bromothymol blue at 614 nm (0–30 s) of three independent tests at each level under the same factor.

2.3. Effect of H+ on the Absorbance of Bromothymol Blue To determine the sensitivity of bromothymol blue on the Adk activity assay, we measured the response of bromothymol blue to hydrogen ion under the most optimal condition, AB3C1D. The absorbance spectrum of bromothymol blue was scanned from 450 to 800 nm in the presence of various concentrations of HCl. The results showed that the absorbance of bromothymol blue at 614 nm gradually declined with the increase in hydrogen ion concentration (Figure2b). The absorbance change can be linearly ﬁtted as the function of hydrogen ion concentration with the following equation (Figure2c): y = 0.138 ∗ x − 0.066

The results suggested that the absorbance of bromothymol blue at 614 nm had a good response to pH change in the assay, and the absorbance change was positively correlated with the hydrogen ion concentration. Molecules 2019, 24, x 6 of 10 Molecules 2019, 24, 663 6 of 10 Molecules 2019, 24, x 6 of 10

Figure 2. The maximum absorption wavelength of the reaction mixture and its relationship with the hydrogenFigure 2. The ion concentration. maximum absorption (a) The absorption wavelength spectra of the reactionof nine different mixture combinations and its relationship in the presence with the Figure 2. The maximum absorption wavelength of the reaction mixture and its relationship with the ofhydrogen 5 mM MgAC ion concentration.2; (b) effect of ( ahydrogen) The absorption ion concentration spectra of nine on the different absorption combinations of the reaction in the presencesystem; ( ofc) hydrogen ion concentration. (a) The absorption spectra of nine different combinations in the presence the5 mM correlation MgAC2;( bof) effectthe absorbance of hydrogen change ion concentration of bromothymol on the absorption blue at 614 of the nm reaction with hydrogen system; (c )ion the of 5 mM MgAC2; (b) effect of hydrogen ion concentration on the absorption of the reaction system; (c) concentration.correlation of the absorbance change of bromothymol blue at 614 nm with hydrogen ion concentration. the correlation of the absorbance change of bromothymol blue at 614 nm with hydrogen ion concentration. 2.4. Effect of Adk Contents on the Reaction Velocity 2.4. EffectThe effect of Adk of Contents Adk contents on the on Reaction the reaction Velocity veloci velocity ty was determined as described in the Materials and Methods section. The results results showed that, with th thee increase increase in in Adk Adk contents, contents, the the reaction reaction velocity velocity The effect of Adk contents on the reaction velocity was determined as described in the Materials increased, and the reaction time time required required to to reach equilibrium shortened (Figure (Figure 33a).a). In the firstfirst 5 s,s, theand absorbance Methods section. of bromothymol The results blueshowed at 614 that, nm with (Abs the increase) declined in Adk linearly contents, with time;the reaction thus, the velocity slope the absorbance of bromothymol blue at 614 nm (Abs614614) declined linearly with time; thus, the slope increased, and the reaction time required to reach equilibrium shortened (Figure 3a). In the first 5 s, of the reaction in the first first 5 s was defined defined as the initial reaction velocity. The plot in Figure3 3bb showsshows the absorbance of bromothymol blue at 614 nm (Abs614) declined linearly with time; thus, the slope that the absorbance change of bromothymol blue at 614 nm could be linearly fitted fitted as the function of of the reaction in the first 5 s was defined as the initial reaction velocity. The plot in Figure 3b shows Adk contents. that the absorbance change of bromothymol blue at 614 nm could be linearly fitted as the function of Adk contents.

Figure 3. The effect of Adk contents on the assay. (a) Effect of different Adk contents on the assay; Figure 3. The effect of Adk contents on the assay. (a) Effect of different Adk contents on the assay; ( b) (b) the correlation of the absorbance change of bromothymol blue at 614 nm with different Adk contents. the correlation of the absorbance change of bromothymol blue at 614 nm with different Adk contents. Figure 3. The effect of Adk contents on the assay. (a) Effect of different Adk contents on the assay; (b) 2.5. Effect of Temperature and KCl on Adk Activity the correlation of the absorbance change of bromothymol blue at 614 nm with different Adk contents. 2.5. Effect of Temperature and KCl on Adk Activity The effect of temperature on Adk activity was investigated to characterize the thermostability of Adk.2.5. EffectThe The effect of results Temperature of showedtemperature and that KCl on Adk on Adk Adk activity activity Activity was was almost investigated unaffected to characterize under 45 ◦C. the However, thermostability when the of Adk. The results showed that Adk◦ activity was almost unaffected under 45 °C. However, when the temperatureThe effect was of increased temperature to 60 onC, Adk Adk activity quickly was lost investigated its activity (Figure to characterize4a). Adk fromthe thermostability the muscle has of a temperature was increased to 60 °C, Adk quickly◦ lost its activity (Figure 4a). Adk from the muscle half-lifeAdk. The of results 30 min showed in 0.1 N hydrochloricthat Adk activity acid atwas 100 almostC[14 ].unaffected Our results under indicate 45 °C. that However, the thermostability when the has a half-life of 30 min in 0.1 N hydrochloric acid at 100 °C [14]. Our results indicate that the oftemperature Adk from Bombyxwas increased mori (BmAdk) to 60 °C, is Adk much quickly lower thanlost its that activity of Adk (Figure from the 4a). muscle. Adk from the muscle thermostability of Adk from Bombyx mori (BmAdk) is much lower than that of Adk from the muscle. has aAllan half-life Hough of 30 et al.min proved in 0.1 that N hydrochloric KCl can almost acid completely at 100 °C inhibit [14]. myokinaseOur results activity indicate [15 that]. Here, the Allan Hough et al. proved that KCl can almost completely inhibit myokinase activity [15]. Here, thethermostability effect of KCl onof Adk Adk activityfrom Bombyx was assessed mori (BmAdk) with the is assay. much The lower results than showed that of thatAdk low from concentration the muscle. the effect of KCl on Adk activity was assessed with the assay. The results showed that low of KClAllan (<5 mM)Hough had et aal. slight proved inhibitory that KCl effect can almost on Adk completely activity. With inhibit the myokinase increase in KClactivity concentration, [15]. Here, concentration of KCl (<5 mM) had a slight inhibitory effect on Adk activity. With the increase in KCl thethe inhibitoryeffect of effectKCl on of KClAdk on activity Adk activity was becameassessed more with and the more assay. obvious. The Aboutresults 70 showed mM KCl that resulted low concentration, the inhibitory effect of KCl on Adk activity became more and more obvious. About 70 inconcentration 50% loss of Adkof KCl activity (<5 mM) (Figure had4 b).a slight Compared inhibitory with effect the “three-minute” on Adk activity. method With [the15 ],increase the inhibitory in KCl mM KCl resulted in 50% loss of Adk activity (Figure 4b). Compared with the “three-minute” method effectconcentration, of KCl on the Adk inhibitory activity couldeffect of be KCl assessed on Ad morek activity easily became with our more developed and more assay. obvious. About 70 mM KCl resulted in 50% loss of Adk activity (Figure 4b). Compared with the “three-minute” method

Molecules 2019, 24, x 7 of 10

Molecules[15], the2019 inhibitory, 24, 663 effect of KCl on Adk activity could be assessed more easily with our developed7 of 10 assay.

Figure 4. The thermostability of Adk and the inhibition of KCl on Adk activity. (a) Effect of temperature Figure 4. The thermostability of Adk and the inhibition of KCl on Adk activity. (a) Effect of on Adk activity; (b) effect of KCl concentration on Adk activity. temperature on Adk activity; (b) effect of KCl concentration on Adk activity. 3. Materials and Methods 3. Materials and Methods 3.1. Chemicals and Materials 3.1. Chemicals and Materials ATP and AMP were purchased from Aladdin (Shanghai, China) in the form of sodium salt. MagnesiumATP and acetate AMP waswere from purchased Sigma (St.from Louis, Aladdin MO, (Shang USA). Bromothymolhai, China) in bluethe form sodium of sodium salt, glycine, salt. andMagnesium other reagents acetate allwas came from from Sigma Sangon (St. Louis, Biotech MO Corp., USA). (Shanghai, Bromothymol China). bluePlastic sodium cuvettes salt, glycine, were purchasedand other fromreagents Centome all came Corp. from (Chengdu, Sangon China).Biotech Corp. (Shanghai, China). Plastic cuvettes were purchased from Centome Corp. (Chengdu, China). 3.2. Adk Preparation and Concentration Determination 3.2. AdkThe Preparation DNA fragment and Co encodingncentration Adk Determination was obtained from the cDNA library by PCR from the midgut 0 0 0 of BombryxThe DNA mori fragmentstrain Dazao encoding using Adk primer was setsobtained (5 -ATGGCACCGGCCGCTGC-3 from the cDNA library by PCRand from 5 -TTACAAA the midgut 0 GCAGACCGTGCTCTGCTG-3of Bombryx mori strain Dazao using). The primer amplification sets (5′-ATGGCACCGGCCGCTGC-3 product was gel-purified, recovered,′ and 5′ and-TTACAAA inserted intoGCAGACCGTGCTCTGCTG-3 plasmid vectors pSKB2. The′). bacterialThe amplification transformants product containing was error-free gel-purified, inserts recovered, were identified. and Adkinserted was into expressed plasmid in vectorsEscherichia pSKB2. coli TheBL21(DE3) bacterial and transformants purified by Ni-NTAcontaining affinity error-free chromatography inserts were (GEidentified. Healthcare, Adk Chicago,was expressed IL, USA). in TheEscherichia fused polyhistidine coli BL21(DE3) tag wasand cleaved purified by by Prescission Ni-NTA proteaseaffinity (GEchromatography Healthcare, USA)(GE Healthcare, and removed Chicago, as described IL, USA). by The Liu fused et al. polyhistidine [16]. Protein tag concentration was cleaved was by − − determinedPrescission protease using the (GE extinction Healthcare, coefficient USA) of and 12,950 removed M 1·L as·cm described1 at 280 nmby Liu on aet NanoDrop al. [16]. Protein 2000C spectrophotometerconcentration was determined (Thermo Fisher, using Waltham, the extinction MA, USA). coefficient of 12,950 M−1·L·cm−1 at 280 nm on a NanoDrop 2000C spectrophotometer (Thermo Fisher, Waltham, MA, USA). 3.3. Adk Activity Assay 3.3. AdkATP Activity and AMP Assay were used as the substrates in the assay. The reaction mixture (1 mL) was composed of 2 mMATP ATP, and 0.6 AMP mM were AMP, used 0.1 mM as glycine–NaOHthe substrates (pHin the 9.0), assay. 0.093 The mM reaction bromothymol mixture blue, (1 andmL) 5 mMwas MgACcomposed2. The of initial2 mM absorbanceATP, 0.6 mM of theAMP, freshly 0.1 mM prepared glycine–NaOH reaction mixture(pH 9.0), at 0.093 614 nm mM was bromothymol adjusted to 1.05blue, with and approximately5 mM MgAC2. 0.5The M initial NaOH absorbance so that the of absorbance the freshly ofprepared the mixture reaction would mixture not be at changed 614 nm afterwas adjusted the addition to 1.05 of Adk.with approximately The purified Adk 0.5 wasM NaOH exchanged so that into the buffer absorbance A (20 mMof the Tris-HCl, mixture pHwould 7.6, 150not mMbe changed NaCl, 5%after glycerol, the addition and 0.1 of mMAdk. dithiothreitol The purified (DTT))Adk was via exchanged gel filtration. into Thebuffer reaction A (20 mM was triggeredTris-HCl, bypH adding 7.6, 150 15 mMµg AdkNaCl, into 5% theglycerol, mixture. and The 0.1 reactionmM dithiothreitol velocity was (DTT)) defined via asgel the filtration. slope of The the absorbancereaction was change triggered ofbromothymol by adding 15 blueμg Adk at 614 into nm the in mixture. the initial The 30 reaction s, which velocity was recorded was defined on a DU as 800the slope nucleic of acid/protein the absorbance analyzer change (BeckmanCoulter, of bromothymol Brea,blue CA,at 614 USA) nm usingin the a initial 1-cm light30 s, pathwhich plastic was cuvette.recorded For on thea DU control 800 nucleic reaction, acid/protein Adk was replaced analyzer with (BeckmanCoulter, buffer A. All measurements Brea, CA, USA) were using carried a 1-cm out ◦ atlight 25 pathC. Each plastic test cuvette. was replicated For the control at least reaction, three times. Adk was replaced with buffer A. All measurements were carried out at 25 °C. Each test was replicated at least three times. 3.4. Orthogonal Design

The concentration of ATP, AMP, bromothymol blue, and glycine–NaOH buffer is vital for Adk activity assay. Consequently, these factors were considered in the orthogonal design to screen the

Molecules 2019, 24, 663 8 of 10 most optimal conditions for Adk activity. Assuming there were three levels for each factor, and the interaction among the factors were not taken into account, the orthogonal experiment of four factors at three levels was designed.

4. Discussion Adk plays a crucial role in maintaining a balance of cellular energy and nucleic acid metabolism. Human Adk isoenzymes speciﬁcally expressed in organs are regarded as important indicators of organ dysfunction [17] or differentiation stages [18]. The Adk activity assays that have been developed so far are largely dependent on the coupled secondary enzymes hexokinase and glucose-6-phosphate dehydrogenase or adenosine monophosphate deaminase [19]. In addition, the different conditions between the coupled enzymes and Adk affect the application of these assays [14]. Therefore, developing a convenient, rapid, sensitive, reliable, and economic assay for Adk activity is of great signiﬁcance. Here, we developed a spectrophotometric assay for Adk activity using bromothymol blue as a pH indicator without coupled enzymes. The effective range of bromothymol blue is pH 6.0–7.6. Adk is optimally active at pH 7.6 [14]. Hence, Adk was dissolved in pH 7.6 buffer to keep it active. The correspondence of pH and the absorbance of the system are listed in Table5. Here, we set the initial absorbance of 1.05 as the beginning of the reaction, which represented pH 6.8 of the reaction system. When 5 µL buffer was added into the system (995 µL), there was almost no change in the pH of the system. At the same time, the assay system could ensure bromothymol blue had a sensitive and stable response to pH change caused by Adk-catalyzed reaction.

Table 5. Correspondence between pH and the absorbance of bromothymol blue at 614 nm.

pH 6.47 6.54 6.73 6.79 6.85 7.07 7.21 7.28 Abs 0.8145 0.9197 0.9438 1.0375 1.0847 1.2385 1.3850 1.4188

The effects of ATP, AMP, bromothymol blue, and glycine–NaOH buffer on the Adk activity assay were determined by a rational orthogonal design. For a full factorial assay with four factors at three levels, the number of tests is up to 81 (34). However, our rational orthogonal design greatly reduced the unnecessary experiments and effectively achieved significant results (Table2). The orthogonal experimental design is a rational design method for multifactor experiment, which selects representative points from a full factorial assay to represent the overall situation. Therefore, it is highly efficient for the design of multifactor experiments with optimal combination levels. By means of the orthogonal design, we greatly reduced the number of required experiments and achieved significant results with the least number of experiments. The optimal temperature for the growth of Bombyx mori is about 25 ◦C. Adk from Bombyx mori was relatively stable below 45 ◦C, implying its significance on the growth and development of Bombyx mori. The Adk structure suggests that the hydrophobic core packing is important for the stability and activity of Adk [20]. AMP has a strong inhibition effect on the reaction with ADP as the substrate, thus resulting in a rapid decrease in the reaction rate with time [6,19]. Here, we found that high concentration of AMP could inhibit Adk activity, which is in line with a previous report. Slater et al. [21] found that the inhibitory effect of AMP was easily observed even when excessive hexokinase and glucose were present. The inhibition was ascribed to the affinity of ADP to Adk, which is lower than that of AMP with Adk. However, in this study, the substrates were ATP and AMP. Therefore, the inhibition of AMP on Adk activity may be attributed to the noncompetitive inhibition of AMP with respect to ATP [22]. To summarize, we developed a simple and rapid assay to determine Adk activity with bromothymol blue as an indicator instead of coupled enzymes. The assays that have been developed so far require the assistance of other enzymes to convert the reaction product to other detectable signals; thus, they consist of multiple reaction steps and are discontinuous. The assays are not accurate as they are easily subject to errors at each step. However, this new assay only relies on the Molecules 2019, 24, 663 9 of 10 protons produced in the reaction and the corresponding absorbance changes of bromothymol blue in the reaction solution. Small changes in pH can lead to significant changes in the absorbance of bromothymol blue at 614 nm. It does not need the assistance of any other enzyme. Bromothymol blue is less expensive than the coupled enzymes, and the assay can be done in just one step, thus reducing the chance of errors and improving reliability. Compared with the existing assays, it is more simple, sensitive, and precise. In addition, it can be applied to research the activation or inhibition of Adk as it is continuous. However, although this assay is simple and precise, the reaction substrate must be prepared freshly as carbon dioxide in the air may interfere with the assay. ATP spontaneously and slowly hydrolyzes in solution, which can also cause an interference on the assay. Nevertheless, it can be a good alternative to the conventional enzymes-coupled Adk activity assay extensively used in clinical and research laboratories.

Author Contributions: K.S., Y.W., Y.L., C.D., and H.H. conceived and designed the experiments; K.S. and Y.L. performed the experiments; K.S., Y.L., and Y.W. analyzed the data; R.C., T.G., and P.Z. contributed reagents/materials/analysis tools; K.S. and Y.W. wrote the draft; Y.W. and H.H. supervised the research; Q.X. and H.H. revised the manuscript. Funding: This work was supported by the National Natural Science Foundation of China (31572465), the State Key Program of the National Natural Science of China (31530071), Fundamental Research Funds for the Central Universities (XDJK2018B010, XDJK2018C063), start-up grant from Southwest University (SWU112111), Graduate Research and Innovation Project of Chongqing (CYB17069), and the Open Project Program of Chongqing Engineering and Technology Research Center for Novel Silk Materials (silkgczx2016003). Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

Adk Adenylate kinase ATP adenosine 50-triphosphates ADP adenosine 50-diphosphates AMP adenosine 50-monophosphate NADPH reduced nicotinamide adenine dinucleotide phosphate Abs absorbance

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