Formal Report Experiment 3 Enzymes

 

http://www.scribd.com/monica_gamboa/d/58234422-Formal-Report-Experiment-3-Enzymes DETERMINATION OF ENZYMATIC ACTIVITY BY DINITROSALICYLIC COLORIMETRIC METHOD AND pH Esclanda, Verna Mae F., Flores, Mary Camelle C., Galvez, Czarina M., Gamboa, Maureen Allysandra J., Go Imon, Karl Louis L., Gudio, Klaudine Renee T.

ABSTRACT Enzyme activity is affected by several factors. When the enzyme changes in shape and structure, its rate of reaction is varied. Examples of the things that alters the rate of reaction of the enzymatic activity are pH and addition of  chemicals this experiment, dinitrosalicylic acid colorimetric method and changing in absorbance pH were done to determine or thereagents. effects ofInphysical and chemical factors to the invertase activity of sucrose. Negative were observed after they were tested in the UV-Vis Spectrophotometer.

INTRODUCTION Enzymes are prote Enzymes proteinace inaceous ous cataly catalysts, sts, which which speed up the rate of a biochemical reaction. They reduce the activation energy that is essential for starting any type of chemical reaction. With a low energy ener gy requiremen requirementt for activation, activation, the reaction reaction takes place faster. The overall performance of an en enzy zyme me depe depend nds s on va vari riou ous s fact factor ors, s, such such as te temp mper erat atur ure, e, pH pH,, cofa cofact ctor ors, s, ac acti tiva vato tors rs an and d

 “bell” demonstrates the “best” pH suitable for the enzyme enz yme that it rea reache ches s the max maximu imum m reacti reaction on rate of the enzymatic activity. Di Dini nitr tros osal alic icyli ylic c acid acid (D.N (D.N.S .S.A .A.. or 3: 3:55dinitrosalicylic acid) is a (yellow) reagent used to determine sugar content especially glucose. The DNS technique is employed in order to estimate sugar present in the blood, in the cerebrospinal fluid and in other human bodily fluids. This is also

1

inhibitors. Since Sin ce en enzym zymes es are protei proteins, ns, they they are very very sensitive to changes in pH. Each enzyme has its own optimum range for pH where it will be most active. This is the the result of the effect of pH on a combin com binatio ation n of fac factor tors: s: (1) the bindin binding g of the enzyme enzym e to subs substrate, trate, (2) the catalytic catalytic activity of  the enzyme, (3) the ionization of the substrate, and (4) the variation of protein structure.2 The initial rates for many enzymatic reactions exhibit bell-shaped curves as a function of pH as shown in the example below.

effectively used in the handling of requirements fo forr di diab abet etic ic cl clin inic ics s in hos ospi pita tall la labo bora rato tori ries es,, considering that only 10 minutes is enough for the process to take place and the reagents are stable, cheap and easily prepared. The amount of  blood sugar in the blood has metabolic im impl pliica cati tion ons s and and is used used to deter etermi min ne the the presence of blood sugar-related disorders such as hyperg hyp erglyc lycemi emia. a. One good good way to assess assess blood blood sugar level is through the use of dinitrosalicylic acid.3

Fig.2 Chemical structure of 3,5-dinitrosalicylic acid Fig.1 Effect of pH on enzymatic activity

The best explanation for this bell-shaped cu curv rve e is th the e st stab abili ility ty of en enzy zyme mes s duri during ng th the e alteration of pH. Meaning, low pH results to slow reaction rate but too high pH shows the same result because of enzymatic loss. The peak of the

  This met This method hod tests for the presence presence of fre free e carbon car bonyl yl gro group up (C=O), (C=O), the soso-cal called led reduci reducing ng su suga gars rs.. Th This is in invo volv lves es th the e oxid oxidat atio ion n of th the e al alde deh hyde yde fun funct ctio iona nall grou group p prese resen nt in in,, for ex exam ampl ple, e, gl gluc ucos ose e an and d th the e ke keto tone ne func functi tion onal al

 

group in fructose. Simultaneously, ly, 3,5di dini nitr tros osal alic icyl ylic ic acid acid (DNS (DNS)) is re redu duce ced d to 3amin am ino, o,55-ni nitr tros osal alic icyl ylic ic acid acid un unde derr al alka kali line ne conditions:

the calibration curve by enhancing the intensity of the developed color. 4

METHODOLOGY A. Su Suc cros rose Ass ssay ay By Din Dinitro itros salic alicyl ylic ic Colorimetric Method   A series of test tubes were prepared as seen in the table below (Table 1). Three drops of concentrated HCl was added to each each tu tube be.. Th The e tu tube be wa was s mi mixe xed d then then o in incu cuba bate ted d in a 90 C water ater bath ath fo forr 5 minutes. Then, 0.15 ml 0.5 M KOH was added to neutralize the solution. A 0.1 M buff buffer er so solu luti tion on abou aboutt 2. 2.8 8 ml was was th then en added and mixed. Three ml of DNS reagent was put in the mixture. The test tubes tub es wer were e aft afterw erward ard imm immers ersed ed in 95oC water bath for 10 minutes to develop the charac cha racter terist istic ic red-br red-brown own col color. or. The test test tubes were then cooled and their absorbance were measured at 540 nm.

Because dissolved oxygen can interfere with gl gluc ucos ose e oxid oxidat atio ion, n, su sulf lfit ite, e, whic which h it itse self lf is no nott necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen. The above reaction scheme shows that one mole of sugar will react with one mole of 3,5dinitrosalicylic acid. However, it is suspected that there the re are many many sid side e reacti reactions ons,, and the actua actuall reaction stoichiometry is more complicated than that that pr prev evio ious usly ly de desc scri ribe bed. d. Th The e ty type pe of side side re reac acti tion on depe depend nds s on th the e ex exac actt na natu ture re of th the e re redu duci cing ng suga sugars rs.. Diff Differ eren entt re redu duci cing ng suga sugars rs generally yield different color intensities; thus, it is ne nece cess ssar ary y to ca cali libr brat ate e for for ea each ch su suga gar. r. In

 

addition the oxidation the carbonyl in the sugar suto gar, , other oth er side side of reacti rea ctions ons such groups as the decomp dec omposi ositio tion n of sug sugar ar also also compet competes es for the av avai aila labil bilit ity y of 3, 3,55-di dini nitr tros osali alicy cylic lic ac acid id.. As a consequence, carboxymethyl cellulose can affect

Table 1. Test Tube Preparation For Sucrose Assay Using Dinitrosalicylic Colorimetric Method

Test Tube No./ Volume Sucrose Std. Solution (1mg/ml) Distilled Water

Blank

1

2

3

4

5

6

0 ml

0.25 ml

0.50 ml

0.75 ml

1.00 ml

1.25 ml

1.50 ml

1.50 ml

1.25 ml

1.00 ml

0.75 ml

0.50 ml

0.25 ml

0 ml

B. Effect Effect of pH on Invert Invertase ase Activi Activity ty Six numbered test tubes were prepared. 2.9 ml 0.1 M buffer solution with different pH was added in each test tube: Table2. Test Tube Preparation For Effect of pH on Invertase Activity

Test Tube No. pH

1

2

3

4

5

2

3

7

7.5

11

Enzyme stock solution with amount of  0.1 ml was added to each test tube. After th the e te test st tu tube bes s were were mixe mixed, d, th they ey were were o incu in cuba bate ted d in 60 C water bath for 5 minu minute tes. s. One One an and d a ha half lf ml of sucr sucros ose e solu soluti tion on was was ad adde ded d an and d th the e te test st tu tube bes s

were again incubated in 60 oC water bath for another 5 minutes. Three ml of DNS reagent was then added. The test tubes were we re im imme mers rsed ed in 95oC water ater bath ath to deve develo lop p th the e char charac acte teri rist stic ic redred-br brow own n color. col or. The test test tub tubes es were were co coole oled d aft after. er. Blank solutions were prepared by foll follow owin ing g th the e fi firs rstt st step eps s but but in inst stea ead d of  enzyme stock solution, denatured enzyme solution was added. The absorbance was measured at 540 nm.

 

RESULTS AND DISCUSSION   A. Sucr Sucros ose e Assa Assay y Usin Using g Di Dini nitr tros osali alicy cyli lic c Colorimetric Method   In the experiment, as the dinitrosalicylic acid, aci d, a yellow yellow dye, was incorpo incorporat rated ed in the te test st tu tube bes s with with th the e pres presen ence ce of he heat at,, th the e mixt mixtur ure e slow slowly ly tu turn rned ed re redd-br brow own. n. This This is because the conversion of the 3,5dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, which contributed to the red-brown color of th the e mi mixt xtur ure. e. Th The e DNS DNS al also so re reac acte ted d to glucose, a product from invertase activity of  sucrose, and converted to gluconic acid. The absorb abs orbanc ance e of each each hyd hydrol rolyze yzed d sucro sucrose se on testt tubes tes tubes was ide identi ntifie fied d using using the UVUV-vis vis spectrophotometer.

Ta Tabl ble3 e3.. x and and y Va Valu lues es fo forr Am Amou ount nt of Ac Acid id-H -Hyd ydro roly lyze zed d Sucrose and Absorbance at 540nm

Amount of 

Absorbance

Acid-Hydrolyzed Sucrose (x) 0 0.017 0.033 0.050 0.067 0.250 0.100

at 540nm (y) 0 0.004 -0.001 -0.003 -0.003 0.048 0.001

Fig.3 Breakdown of Sucrose to Glucose and Fructose Through Invertase

To comput compute e for the amount amount of acidacidhydrolyzed hydr olyzed sucrose, sucrose, the equat equation ion was used. used. Usi Using ng the formu formula, la, the follow following ing were obtained:

Fig.4 Absorbance vs. Amount of Acid-Hydrolyzed Sucrose

  The line drawn on the graph represented the   “best “best fit fit”” lin line e and wa was s comput computed ed thr throug ough h the lin linear ear regre egres ssio ion n func functi tion on of a scie ient ntiifi fic c calcul cal culator ator.. Th The e slo slopepe-int interc ercept ept for form m com comput puted ed was found to be y=0.215x-0.011. In the graph shown for absorbance vs. amount of acid hydrolyzed sucrose, a linear trend was not identified. This is due to some possible causes. First, inactivity of the sucrose if it was not freshly prepared. Second, the dinitrosalicylic acid might not be reactive. Or, the spectrophotometer was defe defect ctiv ive e or no nott se sens nsit itiv ive e enou enough gh and and ma may y actually commit errors.

B. Effect of pH on Invertase Activity

y=absorbance b=intercept m=slope

 

The intercept was found to be -0.011 while the slope slop e was 0.215. The follo following wing absorban absorbance ce will be us used ed to comp comput ute e fo forr th the e conc concen entr trat atio ion n or amount of acid-hydrolyzed sucrose: Table4. Absorbance (at 540 nm) at certain pH

pH 2 3 7 7.5

Absorbance at 540nm 0.036 -0.004 -0.013 -0.009

11

0.009

Invertase may be used over an extended pH range with an optimum pH at 4.5. This enzyme is fully active from pH 3.0 to 5.5. Use at pH values over 6.0 is not recommended because it causes denaturation of the enzyme. The result of the experimental graph was very far different from the ideal graph. The graph was supposed to be bell ell shaped. But in this experi exp erimen ment, t, the graph graph was U-shap U-shaped. ed. Thi This s Ushaped curve was due to the negative values of  the absorbance. This kind of error was committed because of some possible factors like the pH of  the mixture was n ot accurate or the spectrophotometer itself committed the errors.

REFERENCES 1

PH EFFECT ON ENZYMES. (n.d.). Retrieved January 18, 2011, from http://www.buzzle.com/articles/ph-effect-onenzymes.html 2 ENZYME ACTIVITY. (n.d.). Retrieved January 18, 2011, from http://www.rpi.edu/dept/chem-eng/BiotechEnviron/IMMOB/enzymeac.htm 3 Tan, Sue Ter eres esa a. HOW TO USE DINITROSALICYLIC ACID Retrieved January 18, 2011 http://www.ehow.com/how_5221277_usedinitrosalicylic-acid.html#ixzz1BIa2r4fx Table5. x and y Values for Effect of pH on Invertase Activity

pH (x) 2 3 7 7.5 11

Amount of Acid-Hydrolyzed Sucrose (y) 0.218 0.033 -0.0093 0.0093 0.09

Fig.5 Amount of Acid-Hydrolyzed Sucrose vs. pH

4

Wang, N.S. GLUCOSE ASSAY BY DINITROSALICYLIC COLORIMETRIC METHOD Retrieved January 18, 2011, from

http://www.eng.umd.edu/~nsw/ench485/lab4 a.htm