hello and welcome to the second experiment called the determination of enzymes specific activity the objectives of these experiments are determine the gst enzyme activity and determine the protein concentration by the right method and the last one is determine determine the specific activity of dsd enzyme the fundamental of these experiments are the activity of the gst enzyme in the protein mixtures depends on the amounts of protein used in the reaction therefore it is necessary to know that the protein content in the edit folio so as you can see here on the picture there is a monomer and polymer here so the monomer is a small molecule let's say this monomer contains a two white circle and one black circle while the polymer is is a long chain molecule made up of repeated patterns of monomers for example here there are four uh different uh four repeated patterns of monomer containing white white and a black circle so a monomer is a single molecule that can be joined together with other same molecules to form a polymer the building blocks of the proteins are amino acid which contain elements such as hnoc and more they are the monomers of the proteins when hundreds or thousands of amino acids join together they create the proteins which are then used for many tasks in organisms such as doing work in cells help with that with dna replication and so on so we can say that monomer is an amino acid itself and the polymer is a protein so amino acid there are 20 kinds of amino acid that differentiate by the r chain here there is an amine chain and the carboxyl chain here and the r can be anything so it can be a non-polar it can be uncharted polar or a chart polar so amino acid itself can polymerize to form a linear chains for example like d peptide here at the bottom one so these two amino acids are joined together with the peptide bond here so it can be said that this is a d peptide so moving on so uh how to quantify the protein so and protein quantitative analysis there are three types of it so there is a volumetric and the gastometry and sp spectrum for dometry and the volumetric analysis containing there is a dou haul which is uh the determination of the total end and then formal titration there so they are using the uh alkali metric titration and a gasoline while the gasometric analysis they used uh converting the an amine group to an n gas while the spectrophotometry here so there are two types of spectrophotometry the uv and the visible light the uv is used used for uh quantified proteins with aromatic groups such as phenylalanine thyrosine and riptophan while the visible light such as your buret falling and lorry in the way so in our in our experiment here we are going to use the visible light spectrophotometry we are using the bio-red uh analysis so the bare analysis is measure [Music] the protein complex between the peptide chains uh that making a complex with the buret ion such as co2 plus here and their protein complex uh between the protein and by red reaction here co2 plus will be performed in a blue purple color which is can be measured under the wavelength 550 nanometer so the biored reaction gives a plus reaction to a compound containing ch2 and h2 or chn hn h2 rcs and h2 the advantages of using the biored method is that the concentration of a protein is a comparable to the polypeptide bone and then the method is uh simple and fast all the disadvantages of the bird method here is they are less sensitive and then the color is sometimes an unstable yeah so why this method is less sensitive to others because many a sample that containing like this uh here chains ch2 and several this for this kind of chain so they can be measured by this method so it can be protein it can be others uh sample so that's why it's less sensitive so moving on why a big questions why we need to measure the protein concentration and why we need to measure the enzyme activities is that really important so for our experiment we are going to [Music] measure the gst enzyme so as you can see here in the picture the phase two enzyme reaction there uh involves three different enzymes such as glucoronaceal transferase or ugt in glucoronidation while the second one is enzyme sulfur transferases or st involving in sulfation and the last one is enzyme glutathione as transversal involved in glutathione conjugation so we're going to explain about the dst here or glutathione s transparency this phase 2 enzyme reaction are involved in the excretion of some xenobiotics in our body so including the gst one so as we can see here in the picture the glutathione and enzyme biotic helped by the enzyme of tst will transform or make a glutathione s conjugate to be ex to be excluded from our body for the glutathione conjugation as i already explained to you that there are three enzymes there so the glutathione conjugation uh include and several uh xenobiotics such as electrophilics antibiotics or the synovitics that already uh transform bio biologically to electrophilic trophies while the substrate for the gst itself share three common features such as the the substrate must be hydrophobic and the electrophilic and react to non-enzymatically with potassium at a measurable rate and so moving on to the next slide there are three types of uh gst conjugation or there is three different processes for making the godathion s conjugate or conjugation the first one is a direct conjugation by displacement of an electron uh withdrawal uh withdrawing group so sorry it's covered by this screen here so for example there is a four nitro quinoline one oxide here on the left so this nitro oxide group will be displaced by the gs minus one so that this position will be displaced by the gs while the direct conjugation by adding of glutathione here means that for example the j ethyl malaya here the the h plus ion or the double bond here in the c will be adding uh or will be broken down uh by the gs so the c uh double bond will be replaced by the c with the gs ion here while the conjugation of a strain ring system uh format metabolically for example this is a chlorobenzene or p450 will be a form or uh will be transformed into a 3 4 oxide which is a structurally this is more uh unstable uh by p 450 so that the gs minus or glutathione minus here can be displaced or can be replace the h ion here by the gs ion as you can see here there are several types of gst detoxification or types there is a alpha and fee in mu and tether and fee and zeta and so on and the substrate and the gst enzyme firstly published in 1974 including the cdnb that you are going to use in your experiment so uh moving on uh the preparation upon determination of enzyme specific activity measurement so in this experiment you are going to do uh five different things or five uh continue i mean related uh experiment the first one is prepare the cyto cytosolic fraction containing the gst enzyme from mice this is already prepared by the uh by our staff and the second one is the determination of enzyme activity or gst in a red liver by the cdnb method and the third one is a preparation of provine serum albumin standard curve using the buyer method and the fourth one is determination of the protein content of cytosolic fraction containing gst by the bird method and the last one you're going to determine the enzyme specific activity calculation so overall there are five steps to finally can measure the specific activity of the dst enzyme so for the first preparation cytosolic fraction of the gst from mice if we can read here we have we we need to fit the male rats which is the strain sprocket only one with the weight 200 and up to 250 with the pellet and water until it full and let the red fast for 24 hours prior to the next step then after that you need to sacrifice the red with the neck and leave with the neck dislocation and for the cytosolic fraction preparation containing dst by a centrifuge method so after you did the dislocation so the liver will be uh chop up and then will be uh will be uh separate between the enzyme and the the other fraction by using the centrifuge centrifugation method and then after that the red liver take the red lever and immediately uh put and phosphate buffer uh in the cold one and add ph seven then measure the weight and cross the red liver in a phosphate buffer using a coat blender for five minutes and after that centrifuge the refined homogeneity at 10 000 g for 30 minutes at 44 degrees and take an end transfer the supernatant to a new tube and discharge the precipitated layer and centrifuge the supernatant at 105 g 1000g for 90 minutes at 4 degree it's like a several step of centrifugation to take the tst enzyme and the supernatant layer is cytosolic fraction containing the gst enzyme as i already explained before after the centrifugation there's supposed to be two layers there so here we need to take and out the supernatant and store the cytosolic fraction at minus 80 until for the use for the cdnb conjugation with gsh and the first step that already prepared by our laboratories so the second step is we're going to determine the enzyme activity or gst right liver by the cdnb method cdnp is stand for one chloro 2.4 d nitrobenzene so here as we can see on the table there are two different fraction or two different type one is a dst cytosol fraction one is a an arc or it's a distilled water so why we need to use the distilled water because it's going to be used as a blank here so please prepare two types of of lust there [Music] as you can see on the explanation of the experiment you are going to prepare two flags one is cytosol fraction and then the other one is distilled water and put 920 for split buffer and then put 20 microliter of gsh and 20 microliter of cdnb and then the last one is add the cytocytosol fraction for example and distill water for blank the gsdnb conjugate when when after you mix it up all of this reagent you you will have a gsd and b conjugate product form and then measure at 340 nanometer from minute 0 to minute 3 using a spectrum photometric as a simple kinetic program so it means after you mix all the reagent there so you need to uh measure the zero minute and minute three so you have two different uh measurement results for each sample so you will you will have four different measurement there uh the one two for example and two for plain so please make a note that the measurement time which is considered as the 0 minute the same as the other treatment it means that if you are mixed all the reaction so the zero minute it means the the last time when you edit the cdnb for the sample also similar for the blank one so starting time is calculated yeah after you add at the city and b so the city and b1 should be added the last after you put all the reaction there such as first cytosol fraction phosphate buffer gsh and the last one you need to put the cdnb and make it as zero minute so the end of product will be rate or observation absorbent absorption per minute so after that the gst enzyme activity is then measured in micro mole product per minute so the observation you need to measure there is reaction speed so it means the absorbance the delta absorbance so before uh so as you can see here there is a two uh measurement the zero minute and the minute three so the delta minute it means that the minute three uh minus minute zero so you can got the delta absorbance there per minute and then the other calculation here is you need to calculate the gst activity and speed reaction in b here divided by eight point five hundred uh [Music] micro mole percent per micro mole centimeter so you can got the micro mole gstnb per minute is stated as c the result of this calculation is needed for further calculation the activity of the enzyme so you need to measure all of this step and the third one after you measure the speed of speed of reaction so the third one is making a standard curve why you need to make a standard curve because you need to measure the concentration of the gst enzyme so here is how to measure how to make the bsa standard curve or a bsa stand for the bovine serum albumin using the biored method so the first materials is biored reagent and the standard solution of bsa and the phosphate buffer you can read by yourself and then the procedure is very simple so wait the albumin and put it in a volumetric flask and add little quartz and make an albumin standard curve with the series concentration ranging from 100 to 700 microgram per milliliter so the procedure in c here is very important because you need to make a range of concentration that you're going to use to make a standard curve so here it is the standard curve table here so there is one up to six and the other one as a sample so it's going to be a seven different uh flask or seven different solution you need to prepare the first one is a albumin solution which is contained the bsa one microgram per milliliter and the second plus you is the vsa concentration of 1.5 and the other one is 2 and 3 and 4 and for the last one there is no bsa there because it's going to be your blank and then the last one is your sample of gst sample that you are going to figure out how many uh how many concentration is there so the standard curve will be making this uh six uh six absorbance uh reading and you need after you read the absorbance there so you need to make a linear graph equation uh like this is the concentration this is the absorbance there so you you're going to find out the the concentration of the sample you are added uh you are going to measure so for example uh or after you mix all of the reaction to make the standard curve and then you got the equation like for example y uh y equal to b x plus a and for example if the absorbance is y so if the absorbance is 0.234 then the 0.234 equals bx plus a and the x1 stands for your concentration your sample concentration so it means that 0.234 which is y minus a divided by b so the four step is the determination of protein concentration of cytosol fraction containing the gst so here also uh all you need to do is just mix mix all of the reagent here cytosol fraction phosphate buffer and bioreaction and incubate those solution in the room temperature for 10 minutes and measure the absorbance absorption of the mixed solution at 550 nanometer against a blank which is containing no cytosol fraction and then after you read all of the this absorbance here this this this one this absorbance here you're going to measure the cytosol protein content which is the total uh the total volume per cytosol fraction milligram per microliter and this one is you can get this one by uh measuring this one first and within within the form microliter there were going to be some milligram protein so it means after you you measure the eggs uh protein concentration which is shown here you can put the concentration as a cytosol protein contained by dividing the ten thousand uh the thousand one times the total volume uh divided cytosol fraction volume and then within the four microliter there they are going to be d the cytoso protein contain within a four microliter they're going to be a milligram protein so please make a note the one unit of enzyme is the total of enzyme that catalyze one microliter of substrate reaction per minute and an enzyme specific activity is the total of enzyme units per milli divided by the protein concentration in milligram per milli or the total enzyme unit over milligram protein therefore the unit for specific enzyme activity is unit per milligram and then the purity of enzyme is proportional to the value of its specific activity meaning that the greater value of the specific activity the higher the purity of the enzyme so the next question is why we need to measure the enzyme activities regarding to the pharmaceutical fields why um ensuring the gst enzyme is so important for example here there is a one scientific report here with the title advance in antitumor agents start getting glutathione as transversal so the dst is as already explained to you that yes it's a family office 2 detoxification enzyme so [Music] the this enzyme catalyzed the conjugation of glutathione including the chemotherapetic agents so the tst is an enzyme that will be excluded or catalyze or will be ex will be will will help the excretion of some semibiotics so the gst in a [Music] cancer patient that as you can as you can as you know that the cancer is such a cell which is uh have a super growth that they can uh divide the cell can divide into a multiple or trill multiple uh produce multiple cell so you can imagine that the tst enzyme is often produced there so it means the excretion of some xenobiotics will be totally also uh multiple times ex doing excretion so it means that whenever the cancer patients they receive some chemotherapy so it means the cell will be expressed or will be exclude the chemotherapetics drugs also in huge amounts so the gst enzyme can be can be targeted as an uh xenobiotic target so whenever the gst is inhibited so it means that the chemotheraphatic drug can be used or can be treated the cancer patient successfully because the gst is inhibited and the gst cannot express or exclude any chemotherapeutic drugs outside our body so it means that the chemotheraphatic drugs can can give any benefit a maximum benefit to our body so thank you for listening