Principles

EXERCISE 2. DETERMINING THE OPTIMUM pH FOR TRYPSIN

Introduction

Trypsin is a kind of protease. This enzyme is present in the small intestine and can break down protein into amino acid. Different enzymes may have different optimum pH levels. At the optimum pH, the enzymes work best. The activity is the highest. In lower pH or higher pH, the excessive hydrogen or hydroxide ions may break the ionic bonds. This changes the shape of the enzyme. The shape of the active site also changes. This lowers the catalytic activity.
Photographic film has a protein called gelatin that coats its surface. If it is removed, a whitish stain will appear.

Principles

In this experiment, we place a photographic film strip into a trypsin solution. The protease will digest the gelatin coating and make it whitish. Different buffer solutions will be added to the solutions as well, to change the surrounding pH. The degree of digestion of the gelatin reflects the activity of the enzyme. Therefore, the optimum pH can be estimated.

Procedure

1. 
   Set up the water bath and adjust the temperature to 37°C.
   
2. 
   Pipette 1 mL buffer solution of pH 1.0 into a test tube.
   
3. 
   Add a strip of photographic film into the solution and put it into the water bath for 5 minutes.
   
4. 
   Pipette 1 mL of trypsin solution into another test tube. Put it into the water bath for 5 minutes.
   
5. 
   Pour the 1 mL of trypsin solution into a test tube with film.
   
6. 
   Record the time it takes for the complete disappearance of gelatin on the film.
   
7. 
   Repeat the experiment with buffer solutions of pH around 2.0, 4.0, 6.0, 8.0, and 10.0.
   

Precautions

1. 
   The temperature must be kept constant, since temperature is a factor affecting the enzymatic activity.
   
2. 
   The gelatin coating is easily scratched and damaged, and should be handled with care.
   
3. 
   The enzyme and the film have to be put into the water bath for 5 minutes before mixing. This allows both substrates and enzymes to reach the op- timum temperature before mixing.
   
4. 
   The tubes must be mixed from time to time.
   
5. 
   If the gelatin coat remains for over 1 hour, we can assume the time to reach the optimum pH to be infinity.
   

2-mashq. Tripsin uchun optimum pH topish.

KIRISH. (Nazariy qism)
Tripsin bu fermentlarning bir turi bo`lib, oqsillarni aminokislotalargacha parchalaydi. Har xil enzimlarni pH optimumlari ham har xil bo`ladi. Optimum pH da enzimlar zo`r ishlaydi. Shuningdek, faollik ham yaxshi bo`ladi. Pastroq yoki balandroq pH larda esa haddan tashqari ko`p vodorod yoki vodorod ionlari ularning ion bog`larini uzib tashlashi mumkun. Bu esa enzim tuzulishini o`zgartiradi. Bu esa uning aktiv (faol) markazini ham o`zgarishiga olib keladi. Keyin esa fermentning katalitik aktivligi tushib ketadi.
Fotoplonka yuzasiga “Jelatin” deb atalgan oqsil qoplangan bo`ladi. Agar u o`chib ketsa, oqarib qolgan zanjir ko`rinadi.
Asosiy qoidalar:
Bu tajribada biz film lentasini tripsinli eritmaga solamiz. Proteaza qoplangan jelatinni eritib oqartirib, qo`yadi. Turli xil buffer eritmalari dastlabki pH ni o`zgartirish uchun qo`shiladi. Jelatinning erish darajasi enzimning faolligida aks etadi. Shunday ekan, optimum pH ni taxminan xisoblash mumkun bo`ladi.
ISHNING BORISHI.
Suv hammomini yoqing va 37⁰C ga sozlang.

1. 
   Pipetkada 1 ml 1,0 pH li eritmani sinov probirkasiga quying.
   
2. 
   U eritmaga fotoplonka tasmasini solib, 5 daqiqaga suv hammomiga qo`ying.
   
3. 
   Yana pipetkada 1 ml tripsin eritmasidan o`lchab, boshqa sinov probirkasiga quying va 5 daqiqaga suv hammomiga qo`ying.
   
4. 
   Avvalgi 1 ml li tripsin eritmasini fotoplonkali sinov probirkasiga quying.
   
5. 
   Fotoplonkadagi jelatin ko`rinmay qolgunicha vaqtni sekundomerda kuzating va belgilab qo`ying.
   
6. 
   Tajribani yana 2,0; 4,0; 6,0; 8,0; 10,0 pH li buffer eritmalarda takrorlang.
   

EHTIYOT CHORALARI!

1. 
   Temperatura o`zgarmas saqlanishi kerak, chunki u fermentativ faollik-ka kuchli ta`sir etadi.
   
2. 
   Qoplangan jelatin oson tirnalib ketishi va shikastlanishi sababli, u bilan ehtiyotkorona ishlash tavsiya etiladi.
   
3. 
   Fotoplonka va enzim aralashtirishdan avval 5 daqiqa suv hammomiga qo`yilishi kerak. Bu esa substrat va enzimni optimum holatiga erishishi uchun kerak.
   
4. 
   Vaqti vaqti bilan probirkalar aralashtirib turiladi.
   
5. 
   Agar jelatin qoplamasi 1 soatdan ortiq turib qolsa, biz ferment optimum pH holatiga erishish jarayoni kuzatilmasligini taxmin qilishimiz mumkun bo`ladi.