Research Article Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

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1 Hindawi Publihing Corporation BioMed Reearch International Volume 2016, Article ID , 6 page Reearch Article Azocaein Subtrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Uing Bromelain Sample Diego F. Coêlho, 1 Thai Peron Saturnino, 1 Fernanda Freita Fernande, 1 Pricila Gava Mazzola, 2 Edgar Silveira, 3 and Elia Baile Tambourgi 1 1 Chemical Engineering School, Campina State Univerity (UNICAMP), Avenida Albert Eintein 500, Campina, SP, Brazil 2 Faculty of Pharmaceutical Science, Campina State Univerity (UNICAMP), Rua Sérgio Buarque de Holanda 250, Campina, SP, Brazil 3 Biochemitry and Genetic Intitute, Federal Univerity of Uberlândia (UFU), Avenida Getúlio Varga, 230 Centro, Pato de Mina, MG, Brazil Correpondence hould be addreed to Diego F. Coêlho; dfcoelho@feq.unicamp.br Received 26 November 2015; Accepted 12 January 2016 Academic Editor: Pengjun Shi Copyright 2016 Diego F. Coêlho et al. Thi i an open acce article ditributed under the Creative Common Attribution Licene, which permit unretricted ue, ditribution, and reproduction in any medium, provided the original work i properly cited. Given the importance of proteae worldwide market, the determination of optimum condition and the development of a tandard protocol are critical during election of a reliable method to determine it bioactivity. Thi paper ue quality control theory to validate a modified verion of a method propoed by Charney and Tomarelli in The reult obtained howed that uing azocaein ubtrate bromelain had it optimum at 45 C and ph 9 (Glycine-NaOH 100 mm). We alo quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (72 and mg ml 1 of azocaein, rep.) and a calibration curve that correlate optical denity with the amount of ubtrate digeted. In all analyed ample, we oberved a ignificant decreae in repone after torage (around 17%), which ugget it ue mut be immediately after preparation. Thu, the protocol preented in thi paper offer a ignificant improvement, given that ubjective definition are commonly ued in the literature and thi imple mathematical approach make it clear and concie. 1. Introduction Becaue proteae repreent the larget and mot important egment in the indutrial enzyme market [1], the conolidation of a reliable method to evaluate it quality i obviouly of extreme importance. Thee enzyme are ued in detergent, food proceing, and leather indutry, a biocatalyt in organicynthei,and,amongmanyotherapplication,a therapeutic becaue their role are involved in key deciion throughout an organim in everal phyiological and metabolic procee [2]. The global market for indutrial enzyme i expected to reach US $7.1 billion by 2018 [3] and i traditionally divided into three egment: food, technical, and feed enzyme. In 2000, technical enzyme ued in detergent, leather, textile, andperonalcareindutrieaccountedfor65%[4]ofthetotal ale (approximately US $ billion [5]), while food enzyme, which include enzyme ued in dairy, brewing, wine, and juice, were valued at 25% and feed enzyme (ued in animal feed) contributed with 10%. Nearly 70 year ago, Charney and Tomarelli [6] propoed the ue of an azoprotein (a protein coupled with diazotized aryl amine) for the determination of proteolytic activity. The digetion of a olution with uch protein releae the chromophoric group, which i oluble in trichloroacetic acid and give it a red-orange colour. The method itelf relie on the reaction between the ubtrate and an enzyme under it optimum temperature/ph for a given time. The olution colour intenity, read at 440 nm, i a function of the amount of azoprotein digeted, ince all protein remaining precipitate after the addition of trichloroacetic acid. The method i till one of the mot reliable method to tudy the proteolytic activity of enzyme [7, 8] due it colour

2 2 BioMed Reearch International tability and no need of chromogenic reagent. Beide, the ulphanilamide-azocaein preparation i no longer neceary, ince it i now available widely in the market. However, the available protocol that decribe thoroughly the method till are lacking in preenting the evaluation of it analytical parameter, required for method validation. Thu, thi tudy aim to review and validate the azocaein method to etablih it detection and quantification limit, in addition to reagent torage tability and a quantitative definition of enzymatic activity. 2. Material and Method 2.1. Bromelain Sample and Other Chemical. Bromelain (catalogue B5144) and azocaein (catalogue A2765) obtained from Sigma-Aldrich (St. Loui, USA) were choen a tandard for thee tudie, being ued to prepare tock olution at different ph. Unle pecified, all other reagent were alo obtained from Sigma-Aldrich Subtrate Solution. Given the nature of thi tudy, the amount of powdered ubtrate and buffer ued will depend on the concentration and ph of each experiment. The ubtrate ph and concentration are part of the tudied variable and are decribed in the following method. All ph buffer were prepared following common protocol decribed elewhere [9]. Baically, 4 ml of ethanol i added to the powdered ubtrate in a 120 ml beaker and i tirred uing a magnetic tirrer to olubilie all aggregated protein and i then diluted with 96 ml of appropriated buffer (100 mm) Bromelain Stock Solution. Bromelain tock olution wa prepared following a modified verion of a method decribed by Hale et al. [10]. The 1 mg ml 1 enzyme olution wa prepared uing a 100 mm buffer of different ph (ince it wa alo under invetigation). Concentration wa choen baed on it maximum olubility at experimental condition Enzymatic Aay. The method conit in mixing equal volume of ubtrate and enzymatic ample at a given temperature and ph that correpond to the optimum condition of the enzyme under invetigation. For practical reaon we choe 125 μl,aitimallenoughtoavoidwatingreource and doe not compromie the method preciion. The kinetic of the digetion were tudied during 420 minute uing ubtrate concentration in a range from 0.1 to 3.0% (w/w) in order to determine a uitable time of digetion. The reaction wa terminated adding 750 μl of 5% trichloroacetic acid (TCA) to the enzyme-ubtrate mixture. The coagulated protein wa removed by centrifugation at 2000 gfor10minatroomtemperature.theobtainedupernatant wa then added to a N NaOH olution uing a 1 : 1 (v/v) ratio and it aborbance wa read at 440 nm. The blank wa obtained by mixing the TCA to the ubtratepriortotheenzymeaddition Optimum ph and Temperature for Bromelain. The optimum ph and temperature for aaying bromelain activity were determined by performing a full factorial deign of experiment uing both variable in two level and three central point. The ph ranged from 6 to 8 and temperature from 25 Cto45 C in the factorial deign. Temperature wa kept contant during ubtrate digetion by uing a Techne R Dri-Block R heater, model DB-3D. Thi deign wa extended to a central compoite deign, which had it variable range adjuted baed on the reult of the firt deign. All tatitical data wa generated and analyed uing R [11], coupled with R-Studio [12], and uing package akima [13], DoE.bae [14], ggplot2 [15], and RColorBrewer [16] Calibration Curve. Uing the curve of azocaein digetion obtained previouly (a decribed in the topic Enzymatic Aay), a correlation between the colour intenity and the ubtrate concentration wa created. The principle i imple: if the enzyme diget the ubtrate for enough time, we would achieve the olution maximum colour intenity, ince all chromophoric group had their bond to the protein broken and thu are oluble in TCA. Thi atifie the aumption made in azocaein original protocol [6], which tate that a completely digeted azocaein olution hatheamecolourintenityaanundigetedample. The calibration curve i obtained by plotting the optical denity meaured when the time of digetion wa 420 min and the concentration of ubtrate at t= Detection and Quantification Limit. Thelimitofdetection (LoD) and limit of quantification (LoQ) for the protocol were baed on the tandard deviation of the repone and the lope of the mean of calibration curve, following ICH guideline [17], and are given by the equation below: LoD = LoQ = 3.3 σ, 10 σ, where σ i the tandard deviation of the repone and i the lope of the calibration curve. A decribed by ICH, the reidual tandard deviation of a regreion line can be ued a the tandard deviation during calculation Stability Aay. Stability aay followed the protocol decribed in a document provided by the US Department of Health and Human Service called Guidance for Indutry: Bioanalytical Method Validation [18]. Short-Term Temperature Stability. Three aliquot of each of the low and high concentration were thawed at room temperature, kept for 8 hour, and then analyed. Long-Term Stability. The torage time in a long-term tability wa evaluated within an interval of ix week, time uually neceary to perform a whole batch of our routine experiment. Long-term tability wa determined by toring three aliquot of each of the low and high concentration at 5 C. To (1)

3 BioMed Reearch International 3 ph ph ph Temperature ( C) (a) Temperature ( C) (b) Temperature ( C) (c) Optical denity (ab) Optical denity (ab) Optical denity (ab) Figure 1: Repone contour of condition optimiation for bromelain olution. avoid contamination, each ample wa tored in it own vial andanalyedonixeparateoccaion. Freeze and Thaw Stability. Three aliquot at each of the low and high concentration were tored at 20 Cfor24hour andthawedunaitedatroomtemperature.whencompletely thawed, the ample were refrozen for 24 hour under the ame condition. The freeze-thaw cycle wa repeated two more time and then analyed on the third cycle. 3. Reult and Dicuion 3.1. Optimum Condition. The tudy and determination of bromelain biochemical propertie have been tudied extenively before through everal method but our interet wa to determine the optimum condition pecifically for the ubtrate under invetigation to evaluate it at it bet. Figure 1(a) correpond to reult obtained from the firt experimental deign and how that at uch variable range thepheemtohavenoinfluenceovertheenzymeactivity. Then we modified the experimental deign by increaing the ph range in order to confirm the obervation. However, theenzymehowedomeincreaeinitactivityatbaicph (Figure1(b))andervedtoetablihthevariablerangefor the central compoite deign (CCD) hown in Table 1. Figure 1(c) how clearly that bromelain ha an impreively wide range of ph and temperature that can diget azocaein ubtrate with no apparent lo in it enitivity. It alohowthatbromelainitillactiveatmoderatelyhigh temperature [19]. Due to local operational reaon we choe ph 9 and 45 C a the condition to be ued in the next tep Time (min) Azocaein concentration 0.10% 0.25% 0% 0.75% 0% 0% 2.50% Figure 2: Azocaein digetion curve at 45 CandpH9uing bromelain 1 mg/ml with ubtrate concentration from 0.1 to 3% (w/w). Table 1: Rotational central compoite deign ued to tudy and determine aay optimum condition hown in Figure 1(c). Level Factor Temperature ( C) ph of thi tudy. For thi cae, ph 9 Glycine-NaOH (100 mm) buffer wa ued during ubtrate preparation Calibration Curve. Figure 2 how the kinetic curve obtained for each concentration of azocaein ubtrate ued. A expected, curve with lower ubtrate concentration were completely digeted in a matter of a few minute, while olution at 3%, 2.5%, and 2% eem to be cloer to uch point but the enzymatic reaction would till be in proce. By plotting the azocaein concentration againt it correpondent optical denity for all curve at 420 min and uing the aumption made by Charney and Tomarelli [6] we obtain a calibration curve which create a relationhip between thee two variable (Figure 3). The ubtrate concentration wa converted eaily from ma fraction to mg ml 1 by taking in account the olvent pecific ma and the volume retraction caued by the addition of ethanol. The divergence between curve i mainly due the fact that reaction uing ubtrate at 2.5% and 3.0% eem to have ignificant amount of undigeted ubtrate and thu the aumption become invalid. Therefore, the olid line (SL) curve repreent the data erie without thee point. Reult from tatitical analyi for both curve are preented in Table 2.

4 4 BioMed Reearch International Table 2: Summary of tatitical analyi reult for both curve. Solid line (SL) Dahed line (DL) Coefficient Std. error t-value R 2 Intercept Slope Intercept Slope Azocaein concentration (mg/ml) Figure 3: Calibration curve for azocaein concentration uing 1 20 mg/ml(olid line, SL) and 1 30 mg/ml (dahed line, DL). A the preented data ugget, it i clear that removing the point related to unfinihed reaction put the correlation in a confidence level allowing it to be ued a a calibration curve. Conider C AZO (mg/ml) = Ab. (2) The limit of detection and quantification were calculated uing (1) and their reult are preented below. Data wa converted to mg ml 1 uing (2) and coefficient obtained for SL. Conider LoD = LoQ = 3.3 σ = = 72 mg/ml 10 σ = = mg/ml. 3.3 (6295) (6295) = Ab = Ab Oneunit(U)ofproteolyticactivitywadefinedatheamount of enzyme capable of digeting 1 mg of ubtrate per minute, a given in the equation below: (3) A (U) = C AZO V 2 Total t V ENZ, (4) where C AZO i the concentration of azocaein obtained uing (2); V Total i the um of volume of TCA, ubtrate, and enzyme olution (V ENZ )uedinthedigetionandt i the digetion time (in minute). 1 Azocaein concentration 2 3 Time (day) Figure 4: Short-term tability reult for azocaein ubtrate Stability Aay. Subtrate torage tability i another important feature to be evaluated in order to etablih a protocol. Short-term tability i important to evaluate whether the ubtrate can be kept at room temperature during a daylong et of experiment (Figure 4). Reult of time = 0 are relative to a ubtrate olution right after it wa prepared, while ubequent day howed reult of each ample, taken from the ame tock olution, left for 8 hour at room temperature prior to analyi. Reult how a ignificant lo of ubtrate repone in both concentration (around 10%) when compared to the tock olution but that a imilar variation i oberved within the time interval tudied. Long-term tability i evaluated to check whether a olutioncanbetoredandforhowlong,withoutbeenfrozen. While there wa no oberved formation of inoluble olid in the tock olution during torage, the repone of ubtrate had a ignificant lo (around 17%) after 14 day but then it tabilized (Figure 5). Thi fact doe not eem to create any interference in any tep of the method but ugget that the ubtrate olution would offer a maximum repone when ued right after preparation. Further tudie will be neceary to undertand the phenomena involved in the decreae of repone over time. The decreae in repone for the ubtrate digetion alo occurred during freeze-thaw cycle (ee Figure 6), which reinforce the hypothei that it i not caued by microbial activity but omehow related to the ubtrate olubility. The oberved error were lower than the one oberved during

5 BioMed Reearch International Azocaein concentration Time (day) Figure 5: Long-term tability for azocaein ubtrate tored at 5 C. 1 Azocaein concentration 2 3 Freeze-thaw cycle Figure 6: Subtrate tability after freeze-thaw cycle. long-term and hort-term tudie, which make it the mot uitable option for torage at the moment. 4. Concluion The protocol decribed followed the main guideline preented by ICH and etablihe a reliable procedure to analye biological activity of proteolytic enzyme. Beide, the method ue a ma correlation between the ubtrate ued and the optical denity oberved in the potdigetion ample. Although a imple and obviou idea, it offer a ignificant improvement, given that ubjective definition are commonly ued in the literature. Beide, we ran a erie of tability aay in order to evaluate the ubtrate and oberved that a ignificant lo (10% 20%) occurred in all ubtrate ample, uggeting that ubtrate olution offer an enhanced repone when prepared right after it ue. A the undertanding of the mechanim controlling the lo in ubtrate repone wa not part of thi reearch, further experiment will be performed and analyed eparately. Nomenclature ICH: International Conference on Harmoniation of Technical Requirement for Regitration of Pharmaceutical for Human Ue. Conflict of Interet The author declare that there i no conflict of interet regarding the publication of thi paper. Acknowledgment The author would like to acknowledge the financial upport of FAPESP (São Paulo Reearch Foundation), PROPP-UFU (Dean of Reearch and Graduate Studie at the Federal Univerity of Uberlândia), and CNPq (National Council for Scientific and Technological Development). Thi Project ha been funded by grant from Sao Paulo Reearch Foundation: FAPESP 2011/ and FAPESP 2012/ Reference [1] O. P. Ward, 3.49 proteae, in Comprehenive Biotechnology, M.-Y. Murray, Ed., pp , Academic Pre, Burlington, Ma, USA, 2nd edition, [2] H. R. Maurer, Bromelain: biochemitry, pharmacology and medical ue, Cellular and Molecular Life Science, vol.58,no. 9,pp ,2001. [3] S. Cumming, Global Market for Indutrial Enzyme to Reach Nearly $7.1 Billion by 2018; Detergent Enzyme Market to Record Maximum Growth, BIO030H, PRWeb, 2014, [4] J. R. Cherry and A. L. Fidantef, Directed evolution of indutrial enzyme: an update, Current Opinion in Biotechnology,vol. 14, no. 4, pp , [5] M. Mccoy, Novozyme emerge, Chemical & Engineering New,vol.79,no.8,pp.23 25,2001. [6] J. Charney and R. M. Tomarelli, A colorimetric method for the determination of the proteolytic activity of duodenal juice, The Journal of Biological Chemitry,vol.171,no.2,pp ,1947. [7]N.S.Leite,A.A.B.deLima,J.C.C.Santanaetal., Determination of optimal condition to obtain the bromelain from pineapple plant produced by micropropagation, Brazilian Archive of Biology and Technology, vol.55,no.5,pp , [8] L.F.Domingue,R.Giglioti,K.A.Feitoaetal., Invitroandin vivo evaluation of the activity of pineapple (Anana comou) on Haemonchu contortu in Santa Inê heep, Veterinary Paraitology,vol.197,no.1-2,pp ,2013. [9] C. Mohan, Buffer: A Guide for the Preparation and Ue of Buffer in Biological Sytem, Calbiochem-Behring Corporation, La Jolla, Calif, USA, [10] L. P. Hale, P. K. Greer, C. T. Trinh, and C. L. Jame, Proteinae activity and tability of natural bromelain preparation, International Immunopharmacology,vol.5,no.4,pp ,2005. [11] R Core Team, R: A Language and Environment for Statitical Computing, R Foundation for Statitical Computing, Vienna, Autria, 2015.

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