EXPERIMENT 9  Effect of substrateConcentration on Enzyme Activity

——MichaelisConstant Assay of Sucrose Enzyme

1. Purpose

(1) Know the range of studyingenzyme-catalyzed reaction kinetics.

(2) Master the principle and method ofdeterming Michaelis constant (K_m).

2. Principle

In enzyme-catalyzed reaction, the initial reaction velocity (v)increases according to the increase of substrate concentration ([S]) while thereaction temperature, pH and enzyme concentration are invariableness. At lastthe velocity tends towards a maximum value called V. Mechaelis and Mentenderived an equation according to the relationship between v and [S]:

This is Mechaelis-Menten equation. K_mis Michaelis constant. We can determine K_m by plot which is aderivation of the Mechaelis-Menten equation. There are two methods.

(1) Plot of V against [S]:

The value of K_m is equivalent to the substrateconcentration at which the velocity is equal to half of V. Therefore we candetermine experimentally the reaction velocity v at the situation of differentsubstrate concentrations, and make plot of v against [S]. The [S] is Km whilev=V/2.

(2) Plot of 1/v against 1/[S]

This plot is aderivation of the Mechaelis-Menten equation: 

Which gives a straight line. The slope of the line is equal to K_m/V.The intercept on the y-axis is equal to 1/V, and the intercept on the x-axis isequal to –1/K_m.

The substrate of the method is sucrose. Hydrolyzing sucrose withdifferent concentrations by quantitative sucrose enzyme can get different quantumof glucose and fructose. We can use the amount of the product to account K_mof sucrose enzyme. Glucose and fructose can react with 3,5-dinitryl salicylicacid to give red complex, we can measure absorbance at 520nm.

  1

  V

  m=K_m/V 

     1

 V

 —1/K_m 1/〔S〕

3. Material

(1) Reagent

① Standard glucose solution: Weigh up glucose of 100mg truly,dissolve in a spot of saturated benzoic acid solution (0.3%) , and thentransfer the solution to volumetric flask of 100ml, dilute to the graduationwith saturated benzoic acid solution, shake up to give standard glucosesolution of 1mg/ml, which can keep in a refrigeratory for a long time.

② pH 4.5 acetic acid buffer of 0.1mol/L: Mix 43ml of Na acetate of1mol/L and 57ml of acetic acid of 1mol/L, dilute to 1000ml.

③ 10% sucrose solution at pH4.5: Weigh up 10g of sucrose truly,dissolve in a spot of pH 4.5 acetic acid buffer of 0.1mol/L, and then transferthe solution to v52667788.cn/yishi/olumetric flask of 100ml, dilute to the graduation with thesame buffer.

④ 3,5-dinitryl salicylic acid reagent:

solution 1: Mix 300ml of NaOH solution of 4.5%, 880ml of 3,5-dinitrylsalicylic acid solution of 1% and 255g of KNaC₄O₈·4H₂O.

solution 2: Mix 10g of crystal hydroxybenzene and 22ml of NaOHsolution of 10%, dilute to 100ml with distilled water.

solution 3: Dissolve 6.9g of NaHSO₃ in 64ml of solution2.

Mix solution 3 and solution 1, shake up acutely to give 3,5-dinitrylsalicylic acid solution, use after a week.

⑤ Yeast sucrase solution:

Weigh up 10g of fresh yeast in a mortar, add a spot of silver sandand 10ml~15ml distilled water and pestle. Keep in a refrigeratory after millingand filtrate, add 2~3 times volume icy acetone in the filtrate. Mix round to uniformityand then centrifugalize, wash the precipitate two times by acetone. Give solidpowdery enzyme after vacuum- desiccation, dissolve the enzyme in 100ml ofdistilled water to get enzyme solution. If there is infusibility object, getrid of it by centrifugalization.

The optimal activity of the enzyme solution is 6~12 units. The definitionof activity unit of sucrose enzyme is the amount of the enzyme in suchsituation: React 5 minutes in definite condition and give 1mg of glucose.

(2) Apparatus

① Two conical flasks of 100ml.

② A mortar.

③ Volumetric flasks each of 50ml and 100ml.

④ A centrifuge(4000rpm).

⑤ Eight stoppered test tubes.

⑥ A constant temperature water boiler.

⑦ Pipets: 1.0ml×2, 2.0ml×2, 5.0ml×5.

⑧ A stopwatch.

⑨ 721 type spectroscope.

4. Procedure

(1) Draw calibration curve

Number six test tubes, add reagents as the following table.

Shake up and heat five minutes truly in boiling water, cool threeminutes in tap water, dilute to 25 ml and shake up. Measure absorbance at520nm, tube 0 is the comparer. Draw calibration curve, the abscissa is contentof glucose and the ordinate is absorbance.

(2) Select the concentration of the enzymein terms of activity

Dilute 10% sucrose solution to the solution of 6.5% at pH4.5, take5ml of solution to each of test tubes, heat preservation for 5 minutes at 25℃ water bath synchronously. Add1.0ml of sucrose enzyme solution to one tube, mix up at once, time bystopwatch, after reacting 5min truly, add 5ml of 0.1mol/L NaOH solution to stopthe enzyme reaction. Add 5.0ml of 0.1mol/L NaOH solution to the other tube atfirst, then add 1.0ml of sucrose enzyme solution (this is the compared tube).

Take three clean stoppered test tubes, number them and add 1.0ml of aforementionedreaction solution and 1.0ml of distilled water in each of tube1 and tube2, add2.0ml of distilled water in tube3. Add 3.0ml of 3,5-dinitryl salicylic acidsolution in each of the three tubes. Heat the three tubes in boiling water for5 minutes, then cool in tap water for three minutes, add water to 25ml, mix upand measure absorbance at 520nm, tube 3 is the comparer. The absorbance ofdetermining tube minus the absorbance of compared tube gives a difference. Wecan find corresponding content of glucose from the calibration curve in termsof the difference. The result that the content of glucose multiplies by elevenis the activity of the enzyme solution.

The enzyme catalyzes compounds in the test tubes to glucose, the optimumcontent of which is between 0.4mg and 1.6mg. It must determine after changingthe concentration of sucrose enzyme or the amount of reaction fluid while thecontent of glucose is too high or low.

(3) Substrate concentration effectingenzyme activity—Michaelisconstant assay

Take seven test tubes, add reagents as the following table. Afteradding the sucrose solution and pH4.5 acetic acid buffer, place them at roomtemperature or in a constant temperature water boiler(20℃or25℃) for 5 minutes. Then add 1.0mlof sucrose enzyme solution in test tubes in turn, shake up at once, note thetime. After reacting for 5 minutes truly, add 0.1mol/L NaOH solution on time,shake up at once to stop the reaction.

After reacting, take eight clean stoppered test tubes, add 1.0ml ofthe reaction fluid and 1.0ml of distilled water in the former seven tubes and2.0ml of distilled water in the eighth tube as comparer. Add 3.0ml of dinitrylsalicylic acid solution in each of the eight tubes, place them in the boilingwater bath for 5 minutes and cool with tap water for 3 minutes. Dilute them to25ml, mix up, measure absorbance at 520nm and note them.

Theoperation of control experiment is the same as the determining tubes’.

5. Resultdisposal

We can find corresponding content (mg, the optimum is 0.4mg~1.6mg,otherwise we must adjust the amount of reaction fluid) of reductive saccharumfrom the calibration curve in terms of the absorbance (determining tube minuscomparer). The content multiplying by 11 is the amount of product, and thenaccount [S], 1/[S], v, 1/v, draw the plot of v against [S] and 1/v against1/[S]. Find K_m of the yeasty sucrose enzyme respectively accordingto the two kinetics curves, and compare them.

The compare shows in the following table.

Attention 342 is molecular weight of sucrose.

ExperimentalGuidance

1. Previewrequire

(1) Know the magnitude of catalyzedactivity of enzyme is according to velocity of enzyme-catalyzed reaction, thatis increase of product or decrease of substrate in unit time. There are manyinfluences, mostly such as temperature, pH, concentration of substrate, enzymeconcentration, inhibitors and agonists.

(2) Preview Mechaelis-Menten equation andknow the significance and account method of K_m.

2. Attention

(1) Enzyme and substrate must be heatedpreservation beforehand

(2) The reaction time ---5 minutes be mustexact absolutely.

(3) The content of reductive saccharum insucrose and enzyme solution is very little, so we cannot do the experiment of eliminatingeffect.

3. Advisementafter experiment

(1) What is the physical significance of K_m?Why do we account K_m using the initial velocity of enzyme-catalyzedreaction?

(2) What is the pivotal operation of theexperiment? Why isv it?

實(shí)驗(yàn)九  底物濃度對(duì)酶活性的影響

——蔗糖酶米氏常數(shù)的測(cè)定

一、 目的要求

1. 了解酶促動(dòng)力學(xué)研究的范圍。

2. 以蔗糖酶為例，掌握測(cè)定米氏常數(shù)（K_m值)的原理和方法。

二、 實(shí)驗(yàn)原理

在酶促反應(yīng)中，當(dāng)反應(yīng)體系的溫度、pH和酶濃度恒定時(shí)，反應(yīng)初速度（v)則隨底物濃度[S]的增加而加速，最后達(dá)到極限，稱(chēng)為最大反應(yīng)速度（V)。Michaelis和Menten根據(jù)反應(yīng)速度與底物濃度的這種關(guān)系，推導(dǎo)出如下方程：

此式稱(chēng)為米氏方程，式中K_m稱(chēng)為米氏常數(shù)，按此方程，可用作圖法求出K_m。方法有：

(一) 以v對(duì)[S]作圖

由米氏方程可知，v=V/2時(shí)，K_m=[S]，即米氏常數(shù)值等于反應(yīng)速度達(dá)到最大反應(yīng)速度一半時(shí)所需底物濃度。因此，可測(cè)定一系列不同底物濃度的反應(yīng)速度v，以v對(duì)[S]作圖。當(dāng)v=V_max/2時(shí)，其相應(yīng)底物濃度即為K_m。

(二) 以1/v對(duì)1/[S]作圖

取米氏方程的倒數(shù)式：

 ∴

以1/v對(duì)1/[S]作圖可得一直線，其斜率為Km/V，截距為1/V。若以直線延長(zhǎng)與橫軸相交，則該交點(diǎn)在數(shù)值上等于-1/K_m。

  1

  V

  m=K_m/V 

 1

 V

 —1/K_m 1/〔S〕

本實(shí)驗(yàn)以蔗糖為底物，利用一定量蔗糖酶水解不同濃度蔗糖所形成的產(chǎn)物（葡萄糖和果糖)的量來(lái)計(jì)算蔗糖酶的K_m值。葡萄糖和果糖能與3，5-二硝基水楊酸試劑反應(yīng)，生成桔紅色化合物，可于520nm處比色測(cè)定之。

三、 試驗(yàn)材料

(一) 試劑

1. 標(biāo)準(zhǔn)葡萄糖溶液：準(zhǔn)確稱(chēng)取100mg葡萄糖溶于少量飽和的苯甲酸溶液（0.3%)，再轉(zhuǎn)移到100ml容量瓶中，用飽和苯甲酸溶液稀釋到刻度，混勻，即得濃度為1mg/ml的標(biāo)準(zhǔn)葡萄糖溶液。冰箱貯藏可長(zhǎng)期保存。

2. pH4.5的0.1mol/L醋酸緩沖液： 取1mol/L醋酸鈉溶液43ml及1mol/L醋酸溶液57ml，稀釋至1000ml即得。

3. pH4.5的10%蔗糖溶液：準(zhǔn)確取10g蔗糖溶于少量pH4.5的0.1mol/L醋酸緩沖液，轉(zhuǎn)移到100ml容量瓶中，用同樣緩沖液稀釋到刻度備用。

4. 3，5-二硝基水楊酸試劑：

溶液I： 4.5% NaOH溶液300ml，1% 3，5-二硝基水楊酸溶液880ml及酒石酸鉀鈉(KNaC₄O₆•4H₂O)255g, 三者一起混合均勻。

溶液II：取結(jié)晶酚10g及10% NaOH溶液22ml，加蒸餾水稀釋成100ml，混勻。

溶液III：取6.9g NaHSO₃溶于64ml溶液II中。

將溶液III和溶液I混合，激烈振搖混勻，即得3，5-二硝基水楊酸溶液，放置一周后備用。

5. 酵母蔗糖酶溶液：稱(chēng)取鮮酵母10g于研缽中，加少量細(xì)砂及10ml～15ml蒸餾水研磨。磨細(xì)后置冰箱中，過(guò)濾，濾液加2～3倍體積冷丙酮，混勻后離心，沉淀用丙酮洗兩次，真空干燥得固體粉末狀酶，再溶于100ml蒸餾水，即得酶溶液。若有不溶物可用離心法除去。

該酶液活力以6～12單位為佳。蔗糖酶活力單位的定義為：在一定條件下反應(yīng)5min，每產(chǎn)生1mg葡萄糖所需要的酶量。

(二) 器材

1. 100ml三角燒瓶2只。

2. 研缽1只。

3. 50ml及100ml容量瓶各1只。

4. 離心機(jī)1臺(tái)（4000rpm)。

5. 糖管8支。

6. 恒溫水浴1臺(tái)。

7. 吸量管：1.0ml 2支，2.0ml 2支，5ml 5支。

8. 停表1只。

9. 721型分光光度計(jì)1臺(tái)。

四、 實(shí)驗(yàn)方法

(一) 標(biāo)準(zhǔn)曲線的繪制

取干凈糖管6支，如下表所示添加試劑。

加畢混勻，于沸水中準(zhǔn)確煮5min，取出用自來(lái)水冷卻3min，稀釋至25ml，混勻后以零號(hào)管調(diào)零點(diǎn)，于520nm處測(cè)定吸光度。以葡萄糖含量為橫坐標(biāo)，以吸光度為縱坐標(biāo)畫(huà)圖。

(二) 根據(jù)活力選擇酶濃度

將10%蔗糖溶液稀釋成pH4.5的6.5%的溶液，取此溶液5ml于試管中，共加兩管。將兩管同時(shí)置于25℃水浴中保溫5min，然后向管中加入蔗糖酶溶液1.0ml，立即混勻，同時(shí)用停表計(jì)時(shí)，準(zhǔn)確反應(yīng)5min后，立即加入5ml 0.1mol/L NaOH溶液以終止酶反應(yīng)。另一管先加入5.0ml 0.1mol/L NaOH溶液，再加入蔗糖酶溶液1.0ml（此為對(duì)照管)。

取干凈糖管3支，第1、2管分別加入上述反應(yīng)液各1.0ml及水各1.0ml，第3管加蒸餾水2.0ml，然后各管均加3.0ml二硝基水楊酸溶液。置沸水浴中煮5min，取出后經(jīng)自來(lái)水冷卻3min，加水至25ml，混勻，以第3管調(diào)零點(diǎn)，于520nm處測(cè)吸光度值。以測(cè)定管的吸光度值減去對(duì)照管吸光度值，求得的差值從標(biāo)準(zhǔn)曲線上查得相應(yīng)的葡萄糖含量，并乘以11，即為每1ml酶溶液的活力。

測(cè)定管中的葡萄糖含量以在0.4mg～1.6mg之間為佳，過(guò)高或過(guò)低均應(yīng)適當(dāng)改變蔗糖酶溶液的濃度或反應(yīng)液用量后再測(cè)之。

(三) 底物濃度對(duì)酶促反應(yīng)速度的影響——米氏常數(shù)的測(cè)定

取試管7支，按下表所示加入試劑。

當(dāng)加完蔗糖溶液及pH4.5醋酸緩沖液后，均放置室溫或恒溫水�。�20℃或25℃)保溫5min，再分別依次向各管加入蔗糖酶溶液1.0ml，立即搖勻，記錄時(shí)間。準(zhǔn)確反應(yīng)5min，再加入0.1mol/L的NaOH溶液，立即搖勻，以終止反應(yīng)。

取8支潔凈糖管，前7支分別加入相應(yīng)的上述反應(yīng)液各1.0ml及蒸餾水各1.0ml，第8支糖管加入2.0ml蒸餾水作空白，然后各管均加入3.0ml二硝基水楊酸溶液，沸水浴5min。取出用自來(lái)水冷卻3min，稀釋到25ml，混勻，于520nm處比色測(cè)定并記錄吸光度值。

五、 結(jié)果處理

根據(jù)各測(cè)定管的吸光度值，從標(biāo)準(zhǔn)曲線上查出相應(yīng)的還原糖毫克數(shù)（以在0.4mg～1.6mg范圍內(nèi)為佳，否則應(yīng)調(diào)整反應(yīng)液用量后重新測(cè)定)，再乘以11，即得各管的產(chǎn)物量，然后分別計(jì)算各反應(yīng)管相應(yīng)的[S]、1/[S]、v及1/v，并作出v-[S]及1/v-1/[S]曲線。再根據(jù)所畫(huà)的兩種動(dòng)力學(xué)曲線，分別求出酵母蔗糖酶的K_m值，并加以比較。

注：342為蔗糖分子量

實(shí)驗(yàn)指導(dǎo)

一、預(yù)習(xí)要求

1. 了解酶的催化活性的強(qiáng)弱是以測(cè)定酶促反應(yīng)的速度為依據(jù)的，即以單位時(shí)間內(nèi)產(chǎn)物形成量或底物件減少量等表示之。它受到很多因素的影響，如溫度、pH、底物濃度、酶濃度、激動(dòng)劑及抑制劑等。

2. 預(yù)習(xí)米氏動(dòng)力學(xué)公式，了解米氏常數(shù)（K_m)的意義及求法。

二、注意事項(xiàng)

1. 酶和底物應(yīng)預(yù)先分別保溫?cái)?shù)分鐘。

2. 反應(yīng)時(shí)間5min應(yīng)絕對(duì)準(zhǔn)確。

三、實(shí)驗(yàn)后思考

1. K_m值的物理意義是什么？為什么要用酶促反應(yīng)的初速度計(jì)算K_m值？

2. 本實(shí)驗(yàn)的操作關(guān)鍵是什么？為什么？