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 vwww.med126.com/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?

实验九  底物浓度对酶活性的影响

——蔗糖酶米氏常数的测定

一、 目的要求

1. 了解酶促动力学研究的范围。

2. 以蔗糖酶为例，掌握测定米氏常数（K_m值)的原理和方法。

二、 实验原理

在酶促反应中，当反应体系的温度、pH和酶浓度恒定时，反应初速度（v)则随底物浓度[S]的增加而加速，最后达到极限，称为最大反应速度（V)。Michaelis和Menten根据反应速度与底物浓度的这种关系，推导出如下方程：

此式称为米氏方程，式中K_m称为米氏常数，按此方程，可用作图法求出K_m。方法有：

(一) 以v对[S]作图

由米氏方程可知，v=V/2时，K_m=[S]，即米氏常数值等于反应速度达到最大反应速度一半时所需底物浓度。因此，可测定一系列不同底物浓度的反应速度v，以v对[S]作图。当v=V_max/2时，其相应底物浓度即为K_m。

(二) 以1/v对1/[S]作图

取米氏方程的倒数式：

 ∴

以1/v对1/[S]作图可得一直线，其斜率为Km/V，截距为1/V。若以直线延长与横轴相交，则该交点在数值上等于-1/K_m。

  1

  V

  m=K_m/V 

 1

 V

 —1/K_m 1/〔S〕

本实验以蔗糖为底物，利用一定量蔗糖酶水解不同浓度蔗糖所形成的产物（葡萄糖和果糖)的量来计算蔗糖酶的K_m值。葡萄糖和果糖能与3，5-二硝基水杨酸试剂反应，生成桔红色化合物，可于520nm处比色测定之。

三、 试验材料

(一) 试剂

1. 标准葡萄糖溶液：准确称取100mg葡萄糖溶于少量饱和的苯甲酸溶液（0.3%)，再转移到100ml容量瓶中，用饱和苯甲酸溶液稀释到刻度，混匀，即得浓度为1mg/ml的标准葡萄糖溶液。冰箱贮藏可长期保存。

2. pH4.5的0.1mol/L醋酸缓冲液： 取1mol/L醋酸钠溶液43ml及1mol/L醋酸溶液57ml，稀释至1000ml即得。

3. pH4.5的10%蔗糖溶液：准确取10g蔗糖溶于少量pH4.5的0.1mol/L醋酸缓冲液，转移到100ml容量瓶中，用同样缓冲液稀释到刻度备用。

4. 3，5-二硝基水杨酸试剂：

溶液I： 4.5% NaOH溶液300ml，1% 3，5-二硝基水杨酸溶液880ml及酒石酸钾钠(KNaC₄O₆•4H₂O)255g, 三者一起混合均匀。

溶液II：取结晶酚10g及10% NaOH溶液22ml，加蒸馏水稀释成100ml，混匀。

溶液III：取6.9g NaHSO₃溶于64ml溶液II中。

将溶液III和溶液I混合，激烈振摇混匀，即得3，5-二硝基水杨酸溶液，放置一周后备用。

5. 酵母蔗糖酶溶液：称取鲜酵母10g于研钵中，加少量细砂及10ml～15ml蒸馏水研磨。磨细后置冰箱中，过滤，滤液加2～3倍体积冷丙酮，混匀后离心，沉淀用丙酮洗两次，真空干燥得固体粉末状酶，再溶于100ml蒸馏水，即得酶溶液。若有不溶物可用离心法除去。

该酶液活力以6～12单位为佳。蔗糖酶活力单位的定义为：在一定条件下反应5min，每产生1mg葡萄糖所需要的酶量。

(二) 器材

1. 100ml三角烧瓶2只。

2. 研钵1只。

3. 50ml及100ml容量瓶各1只。

4. 离心机1台（4000rpm)。

5. 糖管8支。

6. 恒温水浴1台。

7. 吸量管：1.0ml 2支，2.0ml 2支，5ml 5支。

8. 停表1只。

9. 721型分光光度计1台。

四、 实验方法

(一) 标准曲线的绘制

取干净糖管6支，如下表所示添加试剂。

加毕混匀，于沸水中准确煮5min，取出用自来水冷却3min，稀释至25ml，混匀后以零号管调零点，于520nm处测定吸光度。以葡萄糖含量为横坐标，以吸光度为纵坐标画图。

(二) 根据活力选择酶浓度

将10%蔗糖溶液稀释成pH4.5的6.5%的溶液，取此溶液5ml于试管中，共加两管。将两管同时置于25℃水浴中保温5min，然后向管中加入蔗糖酶溶液1.0ml，立即混匀，同时用停表计时，准确反应5min后，立即加入5ml 0.1mol/L NaOH溶液以终止酶反应。另一管先加入5.0ml 0.1mol/L NaOH溶液，再加入蔗糖酶溶液1.0ml（此为对照管)。

取干净糖管3支，第1、2管分别加入上述反应液各1.0ml及水各1.0ml，第3管加蒸馏水2.0ml，然后各管均加3.0ml二硝基水杨酸溶液。置沸水浴中煮5min，取出后经自来水冷却3min，加水至25ml，混匀，以第3管调零点，于520nm处测吸光度值。以测定管的吸光度值减去对照管吸光度值，求得的差值从标准曲线上查得相应的葡萄糖含量，并乘以11，即为每1ml酶溶液的活力。

测定管中的葡萄糖含量以在0.4mg～1.6mg之间为佳，过高或过低均应适当改变蔗糖酶溶液的浓度或反应液用量后再测之。

(三) 底物浓度对酶促反应速度的影响——米氏常数的测定

取试管7支，按下表所示加入试剂。

当加完蔗糖溶液及pH4.5醋酸缓冲液后，均放置室温或恒温水浴（20℃或25℃)保温5min，再分别依次向各管加入蔗糖酶溶液1.0ml，立即摇匀，记录时间。准确反应5min，再加入0.1mol/L的NaOH溶液，立即摇匀，以终止反应。

取8支洁净糖管，前7支分别加入相应的上述反应液各1.0ml及蒸馏水各1.0ml，第8支糖管加入2.0ml蒸馏水作空白，然后各管均加入3.0ml二硝基水杨酸溶液，沸水浴5min。取出用自来水冷却3min，稀释到25ml，混匀，于520nm处比色测定并记录吸光度值。

五、 结果处理

根据各测定管的吸光度值，从标准曲线上查出相应的还原糖毫克数（以在0.4mg～1.6mg范围内为佳，否则应调整反应液用量后重新测定)，再乘以11，即得各管的产物量，然后分别计算各反应管相应的[S]、1/[S]、v及1/v，并作出v-[S]及1/v-1/[S]曲线。再根据所画的两种动力学曲线，分别求出酵母蔗糖酶的K_m值，并加以比较。

注：342为蔗糖分子量

实验指导

一、预习要求

1. 了解酶的催化活性的强弱是以测定酶促反应的速度为依据的，即以单位时间内产物形成量或底物件减少量等表示之。它受到很多因素的影响，如温度、pH、底物浓度、酶浓度、激动剂及抑制剂等。

2. 预习米氏动力学公式，了解米氏常数（K_m)的意义及求法。

二、注意事项

1. 酶和底物应预先分别保温数分钟。

2. 反应时间5min应绝对准确。

三、实验后思考

1. K_m值的物理意义是什么？为什么要用酶促反应的初速度计算K_m值？

2. 本实验的操作关键是什么？为什么？