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?

實驗九  底物濃度對酶活性的影響

——蔗糖酶米氏常數的測定

一、 目的要求

1. 了解酶促動力學研究的范圍。

2. 以蔗糖酶為例，掌握測定米氏常數（K_m值)的原理和方法。

二、 實驗原理

在酶促反應中，當反應體系的溫度、pH和酶濃度恒定時，反應初速度（v)則隨底物濃度[S]的增加而加速，最后達到極限，稱為最大反應速度（V)。Michaelis和Menten根據反應速度與底物濃度的這種關系，推導出如下方程：

此式稱為米氏方程，式中K_m稱為米氏常數，按此方程，可用作圖法求出K_m。方法有：

(一) 以v對[S]作圖

由米氏方程可知，v=V/2時，K_m=[S]，即米氏常數值等于反應速度達到最大反應速度一半時所需底物濃度。因此，可測定一系列不同底物濃度的反應速度v，以v對[S]作圖。當v=V_max/2時，其相應底物濃度即為K_m。

(二) 以1/v對1/[S]作圖

取米氏方程的倒數式：

 ∴

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

  1

  V

  m=K_m/V 

 1

 V

 —1/K_m 1/〔S〕

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

三、 試驗材料

(一) 試劑

1. 標準葡萄糖溶液：準確稱取100mg葡萄糖溶于少量飽和的苯甲酸溶液（0.3%)，再轉移到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的標準葡萄糖溶液。冰箱貯藏可長期保存。

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：準確取10g蔗糖溶于少量pH4.5的0.1mol/L醋酸緩沖液，轉移到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：稱取鮮酵母10g于研缽中，加少量細砂及10ml～15ml蒸餾水研磨。磨細后置冰箱中，過濾，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5min，每產生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1臺（4000rpm)。

5. 糖管8支。

6. 恒溫水浴1臺。

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1臺。

四、 實驗方法

(一) 標準曲線的繪制

取干凈糖管6支，如下表所示添加試劑。

加畢混勻，于沸水中準確煮5min，取出用自來水冷卻3min，稀釋至25ml，混勻后以零號管調零點，于520nm處測定吸光度。以葡萄糖含量為橫坐標，以吸光度為縱坐標畫圖。

(二) 根據活力選擇酶濃度

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，同時用停表計時，準確反應5min后，立即加入5ml 0.1mol/L NaOH溶液以終止酶反應。另一管先加入5.0ml 0.1mol/L NaOH溶液，再加入蔗糖酶溶液1.0ml（此為對照管)。

取干凈糖管3支，第1、2管分別加入上述反應液各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，取出后經自來水冷卻3min，加水至25ml，混勻，以第3管調零點，于520nm處測吸光度值。以測定管的吸光度值減去對照管吸光度值，求得的差值從標準曲線上查得相應的葡萄糖含量，并乘以11，即為每1ml酶溶液的活力。

測定管中的葡萄糖含量以在0.4mg～1.6mg之間為佳，過高或過低均應適當改變蔗糖酶溶液的濃度或反應液用量后再測之。

(三) 底物濃度對酶促反應速度的影響——米氏常數的測定

取試管7支，按下表所示加入試劑。

當加完蔗糖溶液及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，記錄時間。準確反應5min，再加入0.1mol/L的NaOH溶液，立即搖勻，以終止反應。

取8支潔凈糖管，前7支分別加入相應的上述反應液各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。取出用自來水冷卻3min，稀釋到25ml，混勻，于520nm處比色測定并記錄吸光度值。

五、 結果處理

根據各測定管的吸光度值，從標準曲線上查出相應的還原糖毫克數（以在0.4mg～1.6mg范圍內為佳，否則應調整反應液用量后重新測定)，再乘以11，即得各管的產物量，然后分別計算各反應管相應的[S]、1/[S]、v及1/v，并作出v-[S]及1/v-1/[S]曲線。再根據所畫的兩種動力學曲線，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

實驗指導

一、預習要求

1. 了解酶的催化活性的強弱是以測定酶促反應的速度為依據的，即以單位時間內產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、激動劑及抑制劑等。

2. 預習米氏動力學公式，了解米氏常數（K_m)的意義及求法。

二、注意事項

1. 酶和底物應預先分別保溫數分鐘。

2. 反應時間5min應絕對準確。

三、實驗后思考

1. K_m值的物理意義是什么？為什么要用酶促反應的初速度計算K_m值？

2. 本實驗的操作關鍵是什么？為什么？