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And we can use that energy to basically power different types of processes that take place inside our body that require those ATP molecules. So on the other hand, a chemical reaction is said to be endergonic and non spontaneous if a delta g is positive. And ATP synthesis is an example of such an endergonic reaction. So we can see that if we know what the gives free energy value is of some particular reaction, we know whether or not that reaction is actually spontaneous. Now, another important fact that you have to know about this quantity gives free energy is gives free energy only depends on the energy, the free energy value of the product and the free energy value of the reactants. So if we know what the free energy of the product is and the free energy of the reactants, all we have to do is subtract the two to find that gives free energy. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
So we can see that if we know what the gives free energy value is of some particular reaction, we know whether or not that reaction is actually spontaneous. Now, another important fact that you have to know about this quantity gives free energy is gives free energy only depends on the energy, the free energy value of the product and the free energy value of the reactants. So if we know what the free energy of the product is and the free energy of the reactants, all we have to do is subtract the two to find that gives free energy. So the pathway that we take when we go from the reactant to the products does not actually determine, does not change what the Gibbs free energy is. It doesn't matter if we take pathway one, two or three when we go from the reactants to products, gives free energy will not actually change. So if we, for example, compare a reaction that has an enzyme and that same reaction that is uncatalyzed does not have an enzyme, the Gibbs free energy in those two reactions will be exactly the same. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
So the pathway that we take when we go from the reactant to the products does not actually determine, does not change what the Gibbs free energy is. It doesn't matter if we take pathway one, two or three when we go from the reactants to products, gives free energy will not actually change. So if we, for example, compare a reaction that has an enzyme and that same reaction that is uncatalyzed does not have an enzyme, the Gibbs free energy in those two reactions will be exactly the same. So a catalyzed and an uncatalyzed reaction will have the same exact Gibbs free energy value. And that leads us to a very important point. Enzymes, when they act on chemical reactions, they do not affect the Gibbs free energy value. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
So a catalyzed and an uncatalyzed reaction will have the same exact Gibbs free energy value. And that leads us to a very important point. Enzymes, when they act on chemical reactions, they do not affect the Gibbs free energy value. They do not change the energy of the reactants, nor they actually change the energy of the products. And that's exactly why the difference, namely the delta g that gives free energy will remain exactly the same when an enzyme is used or when an enzyme is not used. Now, the final thing I'd like to mention about Gibbs free energy is what happens if Gibbs free energy is equal to zero? | Enzymes’ Effect on Activation Energy and Free Energy .txt |
They do not change the energy of the reactants, nor they actually change the energy of the products. And that's exactly why the difference, namely the delta g that gives free energy will remain exactly the same when an enzyme is used or when an enzyme is not used. Now, the final thing I'd like to mention about Gibbs free energy is what happens if Gibbs free energy is equal to zero? Well, if Gibbs free energy is zero, then no energy is being produced in that reaction that actually can be used in any useful way. In fact, when Gibbs free energy is zero, that reaction is said to have reached equilibrium. And at that moment in time, the rate of the four reaction is equal to the rate of the reverse reaction. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
Well, if Gibbs free energy is zero, then no energy is being produced in that reaction that actually can be used in any useful way. In fact, when Gibbs free energy is zero, that reaction is said to have reached equilibrium. And at that moment in time, the rate of the four reaction is equal to the rate of the reverse reaction. So if the Gibbs free energy is zero, the reaction has achieved equilibrium and is said to be neither spontaneous nor non spontaneous. In such a case, the rate of the four reaction going from reactants to products is equal to the rate of the reverse reaction going from products back to reactants. Now let's move on to Activation energy. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
So if the Gibbs free energy is zero, the reaction has achieved equilibrium and is said to be neither spontaneous nor non spontaneous. In such a case, the rate of the four reaction going from reactants to products is equal to the rate of the reverse reaction going from products back to reactants. Now let's move on to Activation energy. So what exactly is the activation energy? Well, any reaction has some activation energy and this is simply the amount of energy that we have to input for the reaction to take place to convert the reactants to the products or in reverse. Now let's suppose we go from reactants to products. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
So what exactly is the activation energy? Well, any reaction has some activation energy and this is simply the amount of energy that we have to input for the reaction to take place to convert the reactants to the products or in reverse. Now let's suppose we go from reactants to products. In this case, our activation energy is simply this quantity here. It's the difference between the energy of the molecule found on this topmost portion of the hill and the energy of that reacting. This is the GIBS free energy given by Delta G with the symbol on top or simply Delta E A, where the A stands for activation. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
In this case, our activation energy is simply this quantity here. It's the difference between the energy of the molecule found on this topmost portion of the hill and the energy of that reacting. This is the GIBS free energy given by Delta G with the symbol on top or simply Delta E A, where the A stands for activation. Now this topmost apex of the hill describes the energy of the transition state of this chemical reaction. And if you, if you recall from organic chemistry, the transition state is not something that exists for a very long time and that's because it has a very high energy value. As can be seen by the following diagram, this apex has the highest energy value in that reaction. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
Now this topmost apex of the hill describes the energy of the transition state of this chemical reaction. And if you, if you recall from organic chemistry, the transition state is not something that exists for a very long time and that's because it has a very high energy value. As can be seen by the following diagram, this apex has the highest energy value in that reaction. And that's precisely why the transition state does not exist for a very long time. And in fact, because it doesn't exist for a very long time, it's unstable. And we can't actually study how the transition state looks like. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
And that's precisely why the transition state does not exist for a very long time. And in fact, because it doesn't exist for a very long time, it's unstable. And we can't actually study how the transition state looks like. We can't isolate it and we can't examine it because it quickly converts into the products. So the activation energy, delta G with that symbol describes the amount of energy that must be supplied to any reaction in order to actually get it going. Now the activation energy describes how quickly a reaction actually takes place. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
We can't isolate it and we can't examine it because it quickly converts into the products. So the activation energy, delta G with that symbol describes the amount of energy that must be supplied to any reaction in order to actually get it going. Now the activation energy describes how quickly a reaction actually takes place. So a reaction can be spontaneous, it can have a negative delta G value, but it can take place very, very slowly. And if a reaction takes place very slowly, what that means is it has a very high activation energy. So activation energy is not the same thing as Gibbs free energy. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
So a reaction can be spontaneous, it can have a negative delta G value, but it can take place very, very slowly. And if a reaction takes place very slowly, what that means is it has a very high activation energy. So activation energy is not the same thing as Gibbs free energy. GIBS free energy basically describes the difference between the energy of the reactants and the products. But activation energy describes how quickly a reaction actually takes place. So Gibbs free energy talks about where that equilibrium will be achieved while Activation energy talks about how quickly that equilibrium will actually be achieved. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
GIBS free energy basically describes the difference between the energy of the reactants and the products. But activation energy describes how quickly a reaction actually takes place. So Gibbs free energy talks about where that equilibrium will be achieved while Activation energy talks about how quickly that equilibrium will actually be achieved. And so once again, as we'll see in more detail in a future lecture, the apex of this curve describes the energy of the transition state. Now, what exactly does the enzyme do and how does the enzyme affect the activation energy? So we said previously that the enzyme does not change the Gibbs free energy of the reaction. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
And so once again, as we'll see in more detail in a future lecture, the apex of this curve describes the energy of the transition state. Now, what exactly does the enzyme do and how does the enzyme affect the activation energy? So we said previously that the enzyme does not change the Gibbs free energy of the reaction. It has no effect on the energy of the reactants and the products. And so their difference, the Delta G is exactly the same. It remains unchanged when the enzyme acts on that chemical reaction. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
It has no effect on the energy of the reactants and the products. And so their difference, the Delta G is exactly the same. It remains unchanged when the enzyme acts on that chemical reaction. But the enzyme does have an effect on the activation energy. In fact, what the enzyme typically does is it actually lowers the energy of that transition state. And by lowering the energy of the transition state, it makes this mountain smaller. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
But the enzyme does have an effect on the activation energy. In fact, what the enzyme typically does is it actually lowers the energy of that transition state. And by lowering the energy of the transition state, it makes this mountain smaller. And so this height will be smaller, and the delta g that gives the activation energy of that reaction will become smaller. And if we decrease the activation energy by essentially stabilizing that transition state, we will speed up the reaction, because ultimately, it's the activation energy, it's the energy barrier that determines the kinetics, the speed, and the rate of that chemical reaction. So enzymes do not affect the equilibrium. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
And so this height will be smaller, and the delta g that gives the activation energy of that reaction will become smaller. And if we decrease the activation energy by essentially stabilizing that transition state, we will speed up the reaction, because ultimately, it's the activation energy, it's the energy barrier that determines the kinetics, the speed, and the rate of that chemical reaction. So enzymes do not affect the equilibrium. They have no effect on the Gibbs free energy of that reaction. The free energy of the reactants and the free energy of the products remains unchanged in any catalyzed reaction. However, what the enzymes do is they stabilize the transition state, lower its energy, they lower the energy of that transition state, and so they decrease the activation energy, and that speeds up that chemical reaction. | Enzymes’ Effect on Activation Energy and Free Energy .txt |
In the past two lectures, we discussed the concept of a gene mutation, and we examined the different types of gene mutations that exist in nature. Now, let's actually summarize our results, and let's begin by looking at the following flowchart. So, this flowchart basically describes the many different types of mutations that can arise in nature. And it also tells tells us the reasons for our gene mutation. Now, a gene mutation is basically a change in the nucleotide sequence on the DNA that is not a result of genetic recombination. Now, basically, there are two reasons for a gene mutation in the first place. | Summary of Gene Mutations .txt |
And it also tells tells us the reasons for our gene mutation. Now, a gene mutation is basically a change in the nucleotide sequence on the DNA that is not a result of genetic recombination. Now, basically, there are two reasons for a gene mutation in the first place. So one of these reasons is a result of an error that might take place in one of the many processes that take place in the body. For example, one process in which an error can basically lead to a g mutation is DNA replication. So, during DNA replication, our DNA polymerase can basically make a mistake and incorrectly base pair a certain nucleotide pair, and that will create our gene mutation. | Summary of Gene Mutations .txt |
So one of these reasons is a result of an error that might take place in one of the many processes that take place in the body. For example, one process in which an error can basically lead to a g mutation is DNA replication. So, during DNA replication, our DNA polymerase can basically make a mistake and incorrectly base pair a certain nucleotide pair, and that will create our gene mutation. So, these types of natural g mutations that take place as a result of one of the many natural processes that exist in the cell is known as a spontaneous mutation. Now, on the other hand, a gene mutation can also take place as a result of one or more physical or chemical agents known as mutagens. So these are basically outside physical or chemical forces that can basically induce our g mutation. | Summary of Gene Mutations .txt |
So, these types of natural g mutations that take place as a result of one of the many natural processes that exist in the cell is known as a spontaneous mutation. Now, on the other hand, a gene mutation can also take place as a result of one or more physical or chemical agents known as mutagens. So these are basically outside physical or chemical forces that can basically induce our g mutation. And one example is UV radiation. So, UV radiation is an outside physical agent that can basically cause harm and create a gene mutation in DNA. So we have induced mutations or spontaneous mutations. | Summary of Gene Mutations .txt |
And one example is UV radiation. So, UV radiation is an outside physical agent that can basically cause harm and create a gene mutation in DNA. So we have induced mutations or spontaneous mutations. So we have two reasons for our mutations to arise. Now, once we form the mutation, there are two categories of mutations. We have insertion and deletions, as well as point mutations, which are also known as base pair mutations or base pair substitutions. | Summary of Gene Mutations .txt |
So we have two reasons for our mutations to arise. Now, once we form the mutation, there are two categories of mutations. We have insertion and deletions, as well as point mutations, which are also known as base pair mutations or base pair substitutions. And let's begin by discussing insertion deletions. So, to insert something or to delete something means we're basically either placing nucleotides into the DNA or we're removing nucleotides from that DNA sequence. Now, an insertion or deletion of a number of nucleotides that is not a multiple of three, that is not a multiple of the codons that exist in the genetic code will cause the reading frame to shift, which will change the amino acid sequence of the polypeptide chain. | Summary of Gene Mutations .txt |
And let's begin by discussing insertion deletions. So, to insert something or to delete something means we're basically either placing nucleotides into the DNA or we're removing nucleotides from that DNA sequence. Now, an insertion or deletion of a number of nucleotides that is not a multiple of three, that is not a multiple of the codons that exist in the genetic code will cause the reading frame to shift, which will change the amino acid sequence of the polypeptide chain. And this is known as a frame shift mutation. So insertion deletions can form frame shift mutations in which the entire reading frame, the entire mRNA sequence, is shifted. And that means that completely new codons will be read by our ribosomes, and a completely new sequence of amino acids will be produced. | Summary of Gene Mutations .txt |
And this is known as a frame shift mutation. So insertion deletions can form frame shift mutations in which the entire reading frame, the entire mRNA sequence, is shifted. And that means that completely new codons will be read by our ribosomes, and a completely new sequence of amino acids will be produced. And this usually leads to non functional proteins. Now, let's take a look at the other type of insertion deletion, known as a non frame shift mutations. So, on the other hand, an insertion or deletion of nucleotides that is a multiple of three will simply insert or remove a number of amino acids equaling to the number of new codons that are added or removed. | Summary of Gene Mutations .txt |
And this usually leads to non functional proteins. Now, let's take a look at the other type of insertion deletion, known as a non frame shift mutations. So, on the other hand, an insertion or deletion of nucleotides that is a multiple of three will simply insert or remove a number of amino acids equaling to the number of new codons that are added or removed. And this is known as a non frame shift mutation because the reading frame is not actually shifted and because the majority of the amino acid in the sequence is actually unchanged. Now, let's move on to the second category known as point mutation. So, a point mutation, also known as a base pair mutation or a base pair substitution, is basically a change of a single nucleotide on that DNA template. | Summary of Gene Mutations .txt |
And this is known as a non frame shift mutation because the reading frame is not actually shifted and because the majority of the amino acid in the sequence is actually unchanged. Now, let's move on to the second category known as point mutation. So, a point mutation, also known as a base pair mutation or a base pair substitution, is basically a change of a single nucleotide on that DNA template. So, if the point mutation does not actually change the amino acid that is produced, and in this case, it is known as a silent mutation. Now, a silent mutation can either arise on the non coding region of the DNA or on the coding region of the DNA. And the reason a silent mutation can arise on the coding region of the DNA is because our genetic code is degenerate. | Summary of Gene Mutations .txt |
So, if the point mutation does not actually change the amino acid that is produced, and in this case, it is known as a silent mutation. Now, a silent mutation can either arise on the non coding region of the DNA or on the coding region of the DNA. And the reason a silent mutation can arise on the coding region of the DNA is because our genetic code is degenerate. And that means more than one codon can basically code for the same exact amino acid. So, if we have a point mutation that causes that forms a new codon, and that new codon still codes for the same exact amino acid, then we know that this is a point mutation because the same exact amino acid sequence will form. On the other hand, a point mutation that changes the codon and the amino acid that is produced is known as a miscense mutation. | Summary of Gene Mutations .txt |
And that means more than one codon can basically code for the same exact amino acid. So, if we have a point mutation that causes that forms a new codon, and that new codon still codes for the same exact amino acid, then we know that this is a point mutation because the same exact amino acid sequence will form. On the other hand, a point mutation that changes the codon and the amino acid that is produced is known as a miscense mutation. So, we have two types of point mutations. They can either be silent, or they can be miss sensed. Now, actually, all point mutations are also non frame shift mutations. | Summary of Gene Mutations .txt |
So, we have two types of point mutations. They can either be silent, or they can be miss sensed. Now, actually, all point mutations are also non frame shift mutations. So let's put that in parentheses. So, non frame shift. So, all point mutations are non frameshift mutations, while insertion deletions can either be frame shift or non frame shift. | Summary of Gene Mutations .txt |
So let's put that in parentheses. So, non frame shift. So, all point mutations are non frameshift mutations, while insertion deletions can either be frame shift or non frame shift. Now, the final type of mutation that I want to briefly discuss is a nonsense mutation. So, a nonsense mutation can arise if as a result of insertion deletions or point mutations. So, both of these categories of mutations contain nonsense mutations. | Summary of Gene Mutations .txt |
Now, the final type of mutation that I want to briefly discuss is a nonsense mutation. So, a nonsense mutation can arise if as a result of insertion deletions or point mutations. So, both of these categories of mutations contain nonsense mutations. So, a nonsense mutation is a mutation when we basically change a codon that codes for an amino acid into a codon that is a stop codon, that terminates our polypeptide chain, and this basically terminates the polypeptide chain prematurely. And this causes or creates a non functional protein. So, basically, this concludes our discussion on genetic mutations. | Summary of Gene Mutations .txt |
So, a nonsense mutation is a mutation when we basically change a codon that codes for an amino acid into a codon that is a stop codon, that terminates our polypeptide chain, and this basically terminates the polypeptide chain prematurely. And this causes or creates a non functional protein. So, basically, this concludes our discussion on genetic mutations. We have two different types of categories. We have insertion deletions and point mutations. Now, insertion deletions can cause frame shift or non frameshift mutations, but point mutations themselves are always is non frameshift mutations. | Summary of Gene Mutations .txt |
Value for our tyrosine will be five five. And once again, as that PH value, the PH value of five five, all these charges will exactly cancel out. And so if we place our amino acid on this line here, it will not move in this direction, nor will it move in this direction. Now, by the way, what happens if I take this molecule and place it, let's say somewhere here, where at this location, when it is to the right of the pi value, this molecule will tend to want to move back in this direction. And that's because anywhere in this region, this molecule will have a net negative charge. And so, because this negative charge will cause it to move this way, it will gravitate towards our pi value. | Isoelectric Focusing and Isoelectric Point (Part II) .txt |
Now, by the way, what happens if I take this molecule and place it, let's say somewhere here, where at this location, when it is to the right of the pi value, this molecule will tend to want to move back in this direction. And that's because anywhere in this region, this molecule will have a net negative charge. And so, because this negative charge will cause it to move this way, it will gravitate towards our pi value. And likewise, if we take this same tyrosine and place it within this region, in this region, it will have a net negative charge. And so, as a result of that electric field, it will gravitate and move towards this pi line. So if we place this here, it will move this way. | Isoelectric Focusing and Isoelectric Point (Part II) .txt |
And likewise, if we take this same tyrosine and place it within this region, in this region, it will have a net negative charge. And so, as a result of that electric field, it will gravitate and move towards this pi line. So if we place this here, it will move this way. If we place this here, it will move in the other direction. And the same thing is true for all these other cases. For example, Glycine. | Isoelectric Focusing and Isoelectric Point (Part II) .txt |
If we place this here, it will move in the other direction. And the same thing is true for all these other cases. For example, Glycine. If we take Glycine and place it here, it will have a net positive charge. So it will move this way until it reaches that point. And if we take Glycine and place it here, it will move this way until it reaches that pi value of five. | Isoelectric Focusing and Isoelectric Point (Part II) .txt |
Now, as we discussed previously, it's the presence of of the catalytic triad inside the active side of Chimetrypsin that actually gives it the power of catalysis, gives it the ability to actually cleave those peptide bonds. So remember, the catalytic triad is basically this collection of three individual residues aspartate HistoGene and Serene, which work together to basically promote the cleavage of those peptide bonds. Now, the question still remains what exactly gives Chimotrypsin its specificity? What gives Chimotrypsin the ability to only Cleave on the carboxyl end of specific amino acids, those amino acids that contain bulky hydrophobic side chain groups? Now, to answer this question, we actually have to study the shape of the active side, the structure of that enzyme. If we examine the active side of that enzyme, we're going to find something called the S one pocket. | Specificity of Serine Proteases.txt |
What gives Chimotrypsin the ability to only Cleave on the carboxyl end of specific amino acids, those amino acids that contain bulky hydrophobic side chain groups? Now, to answer this question, we actually have to study the shape of the active side, the structure of that enzyme. If we examine the active side of that enzyme, we're going to find something called the S one pocket. And the S One pocket in China trypsin is basically that region to which that side chain group will actually move into. And if we examine the shape and structure of the S One pocket, we're going to find that it's relatively long, so relatively deep and mostly hydrophobic, so non polar. And because of that structure of the S one pocket, only those amino acids that contain side chain groups that are long, nonpolar, do not have any charges, will actually be able to fit into that pocket, into the active side, without creating too much electric repulsion. | Specificity of Serine Proteases.txt |
And the S One pocket in China trypsin is basically that region to which that side chain group will actually move into. And if we examine the shape and structure of the S One pocket, we're going to find that it's relatively long, so relatively deep and mostly hydrophobic, so non polar. And because of that structure of the S one pocket, only those amino acids that contain side chain groups that are long, nonpolar, do not have any charges, will actually be able to fit into that pocket, into the active side, without creating too much electric repulsion. So amino acids such as methionine, phenyl, aline, tyrosine and Tryptophan. So once again, it's the catalytic triad in the active side. It's the presence of these three individual amino acids that give climate Trypsin its catalytic power, the ability to actually catalyze the cleavage of those peptide bonds. | Specificity of Serine Proteases.txt |
So amino acids such as methionine, phenyl, aline, tyrosine and Tryptophan. So once again, it's the catalytic triad in the active side. It's the presence of these three individual amino acids that give climate Trypsin its catalytic power, the ability to actually catalyze the cleavage of those peptide bonds. But it's this long and narrow shape and the fact that it's mostly hydrophobic, it's the shape of the S one pocket that actually gives chymetrypsin its specific nature, its specificity to Cleave only on the carboxyl end of specific side chain groups. Now, as we discussed in our study of proteases, we basically said there are many different types of proteases that exist inside our body and inside nature. Now, so far, we focused on Serene proteases and we use Chimetrypsin as the prototypical seren protease. | Specificity of Serine Proteases.txt |
But it's this long and narrow shape and the fact that it's mostly hydrophobic, it's the shape of the S one pocket that actually gives chymetrypsin its specific nature, its specificity to Cleave only on the carboxyl end of specific side chain groups. Now, as we discussed in our study of proteases, we basically said there are many different types of proteases that exist inside our body and inside nature. Now, so far, we focused on Serene proteases and we use Chimetrypsin as the prototypical seren protease. But of course, in our body, in our digestive system, for example, we have many other examples of seren proteases. The question is, what is the mechanism that these other seren proteases actually use to cleave peptide bonds? Well, it turns out other crises also use this same catalytic triad. | Specificity of Serine Proteases.txt |
But of course, in our body, in our digestive system, for example, we have many other examples of seren proteases. The question is, what is the mechanism that these other seren proteases actually use to cleave peptide bonds? Well, it turns out other crises also use this same catalytic triad. And what that means is they also carry out the catalysis process by using the same exact mechanism, namely covalent catalysis and acid based catalysis and two examples of other cum proteases inside our digestive system that also contain the same catalytic triad is Trypsin as well as elastase. So Chimotrypsin is not the only serum protease that utilizes this catalytic triad. In fact, Trypsin and Elastase are two other serum proteases found in our digestive system that use this same exact catalytic triad and therefore the same exact mechanism of catalysis that we spoke about in the previous lecture. | Specificity of Serine Proteases.txt |
And what that means is they also carry out the catalysis process by using the same exact mechanism, namely covalent catalysis and acid based catalysis and two examples of other cum proteases inside our digestive system that also contain the same catalytic triad is Trypsin as well as elastase. So Chimotrypsin is not the only serum protease that utilizes this catalytic triad. In fact, Trypsin and Elastase are two other serum proteases found in our digestive system that use this same exact catalytic triad and therefore the same exact mechanism of catalysis that we spoke about in the previous lecture. Now, the question is, why do we have different types of C and proteases inside our body? And the question is, what exactly differentiates Trypsin elastics and Chimera Trypsin if they have the same exact catalytic triad? Well, remember, it's the catalytic triad that gives the enzyme its catalytic power, that gives the protease the ability to cleave those peptide bonds. | Specificity of Serine Proteases.txt |
Now, the question is, why do we have different types of C and proteases inside our body? And the question is, what exactly differentiates Trypsin elastics and Chimera Trypsin if they have the same exact catalytic triad? Well, remember, it's the catalytic triad that gives the enzyme its catalytic power, that gives the protease the ability to cleave those peptide bonds. But it's the shape of that active side, the s one pocket that determines the specificity of that protease, the type of peptide bond it actually breaks. And so the difference between Chima, Trips and Trypsin and Elastase is not in the type of catalytic triad used, but it's in the shape of that particular s one pocket. The structure of that s one pocket, as we'll see in just a moment, as a result of a slight variation in the s one pocket of Trypsin and Elastase, we see that these other CM proteases cleave other amino acids. | Specificity of Serine Proteases.txt |
But it's the shape of that active side, the s one pocket that determines the specificity of that protease, the type of peptide bond it actually breaks. And so the difference between Chima, Trips and Trypsin and Elastase is not in the type of catalytic triad used, but it's in the shape of that particular s one pocket. The structure of that s one pocket, as we'll see in just a moment, as a result of a slight variation in the s one pocket of Trypsin and Elastase, we see that these other CM proteases cleave other amino acids. So although Trypsin elastics use the same mechanism, so covalent catalysis and acid based catalysis, which we spoke about in the previous lecture, they differ in their specificity. And that has to do to the fact that there is a slight structural difference in the s one pocket in Trypsin as well as elastics. And let's see what these differences are and what they result in. | Specificity of Serine Proteases.txt |
So although Trypsin elastics use the same mechanism, so covalent catalysis and acid based catalysis, which we spoke about in the previous lecture, they differ in their specificity. And that has to do to the fact that there is a slight structural difference in the s one pocket in Trypsin as well as elastics. And let's see what these differences are and what they result in. So let's begin with trypsin. So Trypsin catalyzes the cleavage of peptide bonds on the carboxyl end of Lysine and Arginine. And if you recall, Lysine and Arginine both contain positive charges on their sidechain groups. | Specificity of Serine Proteases.txt |
So let's begin with trypsin. So Trypsin catalyzes the cleavage of peptide bonds on the carboxyl end of Lysine and Arginine. And if you recall, Lysine and Arginine both contain positive charges on their sidechain groups. Now, the question is, why is this true? So Trypsin uses the same exact catalytic triad that climate Trypsin uses. But what gives this Trypsin the difference in specificity? | Specificity of Serine Proteases.txt |
Now, the question is, why is this true? So Trypsin uses the same exact catalytic triad that climate Trypsin uses. But what gives this Trypsin the difference in specificity? Well, if we examine the s one pocket of Trypsin at the bottom of that s one pocket, we're going to see a residue that we don't see in the s one pocket of Chimetrypsin at the bottom of the Trips. In s one pocket, we have a negatively charged side chain that came from the aspartate that we see in Trypsin and we don't see in Chimetrypsin. So as a result of the negatively charged side chain of Aspartade 189 that is found at the bottom of the s one Trypsin pocket, we see that this Trypsin only Cleaves at the carboxyl end of those amino acids which are long and contain a positive charge at the end. | Specificity of Serine Proteases.txt |
Well, if we examine the s one pocket of Trypsin at the bottom of that s one pocket, we're going to see a residue that we don't see in the s one pocket of Chimetrypsin at the bottom of the Trips. In s one pocket, we have a negatively charged side chain that came from the aspartate that we see in Trypsin and we don't see in Chimetrypsin. So as a result of the negatively charged side chain of Aspartade 189 that is found at the bottom of the s one Trypsin pocket, we see that this Trypsin only Cleaves at the carboxyl end of those amino acids which are long and contain a positive charge at the end. And these happen to be Lysine and Arginine. So if we examine the following hypothetical polypeptide, we have glycine this one here. We have Lysine this one here. | Specificity of Serine Proteases.txt |
And these happen to be Lysine and Arginine. So if we examine the following hypothetical polypeptide, we have glycine this one here. We have Lysine this one here. We have glycine this one. We have Arginine this one and we have glycine, this one. Now, the only ones that contain positive charges are these two amino acids. | Specificity of Serine Proteases.txt |
We have glycine this one. We have Arginine this one and we have glycine, this one. Now, the only ones that contain positive charges are these two amino acids. And only these amino acids will be able to actually fit into the pocket of Trypsin, and at the end will be able to actually interact in a stabilizing manner. So the positive charges of these two side chain groups will interact with the negative charges of this aspartate 189 and that will create a stabilizing effect, it will neutralize the net charge in that space and that will create a very stable effect. So at the bottom of the active cytotrypsin is an aspartate residue and the negative charge of the aspartate side chain group will stabilize those amino acids that contain side chain groups with positive charges, namely the Lysine and the arginine. | Specificity of Serine Proteases.txt |
And only these amino acids will be able to actually fit into the pocket of Trypsin, and at the end will be able to actually interact in a stabilizing manner. So the positive charges of these two side chain groups will interact with the negative charges of this aspartate 189 and that will create a stabilizing effect, it will neutralize the net charge in that space and that will create a very stable effect. So at the bottom of the active cytotrypsin is an aspartate residue and the negative charge of the aspartate side chain group will stabilize those amino acids that contain side chain groups with positive charges, namely the Lysine and the arginine. And so the only bonds that Trypson will be able to cleave are this bond and this bond here. So at the carboxyl end of Lysine and arginine, so we see that a very tiny variation in the structure of the s one pocket in Trypsin gives Trypsin a different specificity than that of chimetrypsin. And finally, let's move on to elastics. | Specificity of Serine Proteases.txt |
And so the only bonds that Trypson will be able to cleave are this bond and this bond here. So at the carboxyl end of Lysine and arginine, so we see that a very tiny variation in the structure of the s one pocket in Trypsin gives Trypsin a different specificity than that of chimetrypsin. And finally, let's move on to elastics. So, if we examine the s one pocket of Elastanes, we'll see the presence of two additional Valene and valine, if you recall, are these small or valine molecules, have these small, relatively small hydrophobic chains. And notice where these two valleys are positions, they're positioned opposite of each other and they essentially play the role of blocking the majority of the bottom portion of that s one pocket. And so what that means is when the side chain group of the amino acid moves into the s one pocket, it cannot actually occupy this space here because this blocks, as a result of the stair kindle of the hydrophobic properties of these side chain groups. | Specificity of Serine Proteases.txt |
So, if we examine the s one pocket of Elastanes, we'll see the presence of two additional Valene and valine, if you recall, are these small or valine molecules, have these small, relatively small hydrophobic chains. And notice where these two valleys are positions, they're positioned opposite of each other and they essentially play the role of blocking the majority of the bottom portion of that s one pocket. And so what that means is when the side chain group of the amino acid moves into the s one pocket, it cannot actually occupy this space here because this blocks, as a result of the stair kindle of the hydrophobic properties of these side chain groups. And so only those amino acids that have relatively small side chain groups and which are nonpolar so uncharged, will be able to fit into this pocket. And so we see that Elastase cleaves peptide bonds on the carboxyl end of small hydrophobic amino acids such as glycine, vallene, Alanine, Valine, Alanine Leucine and Isolucine, as well as serene. So if we have this hypothetical polypeptide chain, we have glycine, lysine, Alanine, phenylalanine, glycine. | Specificity of Serine Proteases.txt |
And so only those amino acids that have relatively small side chain groups and which are nonpolar so uncharged, will be able to fit into this pocket. And so we see that Elastase cleaves peptide bonds on the carboxyl end of small hydrophobic amino acids such as glycine, vallene, Alanine, Valine, Alanine Leucine and Isolucine, as well as serene. So if we have this hypothetical polypeptide chain, we have glycine, lysine, Alanine, phenylalanine, glycine. So the only ones which have a small hydrophobic side chain are glycine Alamine, as well as glycine at the end. So the only two peptide bonds that are going to be cleaved are this peptide bond. So on the carboxyl end of glycine, as well as this one on the carboxyl end of Alanine. | Specificity of Serine Proteases.txt |
So the only ones which have a small hydrophobic side chain are glycine Alamine, as well as glycine at the end. So the only two peptide bonds that are going to be cleaved are this peptide bond. So on the carboxyl end of glycine, as well as this one on the carboxyl end of Alanine. So Lysine and Phenylalanine are not small ones, this one has a large long one and it's positively charged. Although this one is non polar hydrophobic, it's too large to actually fit into this pocket because these two valleyses block the majority of that pocket. And even though this is a glycine, there's no bond on the carboxyl side. | Specificity of Serine Proteases.txt |
So Lysine and Phenylalanine are not small ones, this one has a large long one and it's positively charged. Although this one is non polar hydrophobic, it's too large to actually fit into this pocket because these two valleyses block the majority of that pocket. And even though this is a glycine, there's no bond on the carboxyl side. And so nothing will be cleaved on this side by elastase and so we see that two Vallene residues found in the S One pocket of Elastase block off the majority of the pocket in Elastase. And this allows Elastase to only cleave small hydrophobic residues. And so we see that the majority of the seagram protease is found inside our body. | Specificity of Serine Proteases.txt |
And so nothing will be cleaved on this side by elastase and so we see that two Vallene residues found in the S One pocket of Elastase block off the majority of the pocket in Elastase. And this allows Elastase to only cleave small hydrophobic residues. And so we see that the majority of the seagram protease is found inside our body. For instance, chimetrypsin trypsin as well as Elastase. Although they use the same catalytic triad to basically catalyze the cleavage of the peptide bonds, they actually differ in the types of amino acids that they cleave. Types of peptide bonds that they cleave. | Specificity of Serine Proteases.txt |
On the contrary, it's very much a fluidlike structure. In fact, the viscosity of the membrane is very much like olive oil. It's about 100 times more viscous than water. Now, the question is, why is this the case? Well, because of phenomenon known as lateral diffusion. And what that means is the molecules that constitute the phospholipids and in many cases, the proteins that constitute the membrane are actually in a constant state of horizontal lateral emotion. | Lateral Diffusion of Lipids and Proteins .txt |
Now, the question is, why is this the case? Well, because of phenomenon known as lateral diffusion. And what that means is the molecules that constitute the phospholipids and in many cases, the proteins that constitute the membrane are actually in a constant state of horizontal lateral emotion. And to visualize the lateral diffusion of these lipid molecules or proteins within our cell membrane, we can basically use a technique, a process known as fluorescence recovery after photo bleaching, which basically is also known as FRAP. So what we basically do is we take the membrane and we attach these fluorescent molecules onto the membrane. And what these fluorescent markers will basically help us do is help us visualize the movement of these molecules, the lipids and the proteins within our membrane. | Lateral Diffusion of Lipids and Proteins .txt |
And to visualize the lateral diffusion of these lipid molecules or proteins within our cell membrane, we can basically use a technique, a process known as fluorescence recovery after photo bleaching, which basically is also known as FRAP. So what we basically do is we take the membrane and we attach these fluorescent molecules onto the membrane. And what these fluorescent markers will basically help us do is help us visualize the movement of these molecules, the lipids and the proteins within our membrane. So that's step one. So at a time of zero, the membrane is labeled with fluorescent molecules. Now, let's suppose at time of T one, what we basically do is we choose a certain area, a certain region of that particular membrane. | Lateral Diffusion of Lipids and Proteins .txt |
So that's step one. So at a time of zero, the membrane is labeled with fluorescent molecules. Now, let's suppose at time of T one, what we basically do is we choose a certain area, a certain region of that particular membrane. Let's suppose the area is about 3 squared. And what we do is we direct electromagnetic radiation and electromagnetic pulse. For example, a high intensity laser pulse. | Lateral Diffusion of Lipids and Proteins .txt |
Let's suppose the area is about 3 squared. And what we do is we direct electromagnetic radiation and electromagnetic pulse. For example, a high intensity laser pulse. We direct it exactly at that 3 μm squared area. And what that does is it basically bleaches that area. It destroys those fluorescent molecules. | Lateral Diffusion of Lipids and Proteins .txt |
We direct it exactly at that 3 μm squared area. And what that does is it basically bleaches that area. It destroys those fluorescent molecules. And now, if we examine those molecules, they will not essentially display that color. So at a time of T one, a region of the membrane is bleached with electromagnetic with an electromagnetic pulse, which destroys those fluorescent molecules in that region. So all these molecules here are basically the unbleeached molecules, the ones that weren't destroyed by the electromagnetic radiation. | Lateral Diffusion of Lipids and Proteins .txt |
And now, if we examine those molecules, they will not essentially display that color. So at a time of T one, a region of the membrane is bleached with electromagnetic with an electromagnetic pulse, which destroys those fluorescent molecules in that region. So all these molecules here are basically the unbleeached molecules, the ones that weren't destroyed by the electromagnetic radiation. But these molecules have been bleached. They have been destroyed. The fluorescent markers have been destroyed. | Lateral Diffusion of Lipids and Proteins .txt |
But these molecules have been bleached. They have been destroyed. The fluorescent markers have been destroyed. Now, what exactly are we going to see? Well, if there was no movement and if the membrane was in fact a rigid and a static structure, then that means this area will remain bleached. But what happened is, because there is a lateral movement of these molecules, eventually these bleached molecules will disperse throughout that membrane. | Lateral Diffusion of Lipids and Proteins .txt |
Now, what exactly are we going to see? Well, if there was no movement and if the membrane was in fact a rigid and a static structure, then that means this area will remain bleached. But what happened is, because there is a lateral movement of these molecules, eventually these bleached molecules will disperse throughout that membrane. And what that means is, because these unbleached molecules will replace the bleached molecules in this area, that will recover the fluorescence in that particular area. So over time, the lateral movement of the lipids will disperse the non fluorescent, the bleached molecules. This will return that fluorescence to that initially bleached area. | Lateral Diffusion of Lipids and Proteins .txt |
And what that means is, because these unbleached molecules will replace the bleached molecules in this area, that will recover the fluorescence in that particular area. So over time, the lateral movement of the lipids will disperse the non fluorescent, the bleached molecules. This will return that fluorescence to that initially bleached area. So initially, this is what we see once we bleach it, we see this purple spots, let's say it's purple. And then over time, this purple spot disappears because these bleached purple molecules essentially move away. And so the fluorescence actually is recovered. | Lateral Diffusion of Lipids and Proteins .txt |
So initially, this is what we see once we bleach it, we see this purple spots, let's say it's purple. And then over time, this purple spot disappears because these bleached purple molecules essentially move away. And so the fluorescence actually is recovered. And this can be summarized in the following graph. So the y axis is the intensity, the fluorescent intensity. The x axis is the time, let's say, given in seconds. | Lateral Diffusion of Lipids and Proteins .txt |
And this can be summarized in the following graph. So the y axis is the intensity, the fluorescent intensity. The x axis is the time, let's say, given in seconds. And this is basically what the curve describes. So this straight line is part A before we actually Bleached. This is the point at which we bleach. | Lateral Diffusion of Lipids and Proteins .txt |
And this is basically what the curve describes. So this straight line is part A before we actually Bleached. This is the point at which we bleach. And notice that at that particular section, we no longer see that same type of color, but over time, we see that the color basically returns because of lateral diffusion along that membrane. So this graph demonstrates the recovery of fluorescence in that Bleached area. It confirms the existence of this phenomenon we call lateral diffusion. | Lateral Diffusion of Lipids and Proteins .txt |
And notice that at that particular section, we no longer see that same type of color, but over time, we see that the color basically returns because of lateral diffusion along that membrane. So this graph demonstrates the recovery of fluorescence in that Bleached area. It confirms the existence of this phenomenon we call lateral diffusion. Now, the question is, what determines the rate at which that area actually recovers its fluorescence? So what determines the slope of this particular line? Because the slope is ultimately the rate of recovery of fluorescence. | Lateral Diffusion of Lipids and Proteins .txt |
Now, the question is, what determines the rate at which that area actually recovers its fluorescence? So what determines the slope of this particular line? Because the slope is ultimately the rate of recovery of fluorescence. Well, basically, it's the speed at which these molecules can actually move along that membrane. So the higher the speed, the greater the likelihood that the molecules will move away. And so the higher the recovery rate is. | Lateral Diffusion of Lipids and Proteins .txt |
Well, basically, it's the speed at which these molecules can actually move along that membrane. So the higher the speed, the greater the likelihood that the molecules will move away. And so the higher the recovery rate is. So it turns out that phospholipids in general can move at a rate of about 1 μm squared per second. So that basically means every single second, that phospholipid can move 1 μm along that membrane. So, once again, these phospholipids generally move with a uniform constant value at a rate of about 1 μm squared per second. | Lateral Diffusion of Lipids and Proteins .txt |
So it turns out that phospholipids in general can move at a rate of about 1 μm squared per second. So that basically means every single second, that phospholipid can move 1 μm along that membrane. So, once again, these phospholipids generally move with a uniform constant value at a rate of about 1 μm squared per second. Now, what about proteins? Well, unlike these phospholipids, which basically move at a relatively uniform rate of 1 μm squared per second, proteins basically vary in the rate of movement. Some are immobile, they move very slowly, while others basically move very quickly and so are mobile. | Lateral Diffusion of Lipids and Proteins .txt |
Now, what about proteins? Well, unlike these phospholipids, which basically move at a relatively uniform rate of 1 μm squared per second, proteins basically vary in the rate of movement. Some are immobile, they move very slowly, while others basically move very quickly and so are mobile. Now, what determines the mobility of these proteins? Well, basically, it's the functionality of that protein is the protein attached onto some other component, onto some other structure. Now, for instance, if we look at Rhodopsin, which is basically a photopigment that is found in the retina cells of our body, rhodopsin's functionality depends on its ability to move quickly along the membrane. | Lateral Diffusion of Lipids and Proteins .txt |
Now, what determines the mobility of these proteins? Well, basically, it's the functionality of that protein is the protein attached onto some other component, onto some other structure. Now, for instance, if we look at Rhodopsin, which is basically a photopigment that is found in the retina cells of our body, rhodopsin's functionality depends on its ability to move quickly along the membrane. And so proteins, membrane proteins such as Rhodopsin can actually move at a relatively high rate, which is about the rate of half this quantity. Here, on the other hand, other proteins, because their functionality depends that they basically remain in that single position. In these cases, proteins are relatively mobile. | Lateral Diffusion of Lipids and Proteins .txt |
And so proteins, membrane proteins such as Rhodopsin can actually move at a relatively high rate, which is about the rate of half this quantity. Here, on the other hand, other proteins, because their functionality depends that they basically remain in that single position. In these cases, proteins are relatively mobile. And one example is fibronectin. So Fibronectin is basically a peripheral glycoprotein that is actually attached onto a transmembrane protein known as Integrin, or Integrin. Now, fibronistin is not only anchored onto the Integrin, but the fibronectin is actually itself anchored onto the collagen fibers found in the extracellular matrix. | Lateral Diffusion of Lipids and Proteins .txt |
And one example is fibronectin. So Fibronectin is basically a peripheral glycoprotein that is actually attached onto a transmembrane protein known as Integrin, or Integrin. Now, fibronistin is not only anchored onto the Integrin, but the fibronectin is actually itself anchored onto the collagen fibers found in the extracellular matrix. On top of that, the Integrin is actually also attached onto the actin filaments that are found inside the cytoplasma, inside the cytoskelet and the cytoplasm. So, because of all these anchoring positions and attachment points, fibro nectin doesn't actually move. It moves very slowly. | Lateral Diffusion of Lipids and Proteins .txt |
On top of that, the Integrin is actually also attached onto the actin filaments that are found inside the cytoplasma, inside the cytoskelet and the cytoplasm. So, because of all these anchoring positions and attachment points, fibro nectin doesn't actually move. It moves very slowly. It's essentially immobile. So we conclude that the cell membrane is not a static rigid structure. It's very fluid like. | Lateral Diffusion of Lipids and Proteins .txt |
So in 1958, two individuals, one by the name of Matthew Methleton and the other one by the name of Franklin Stahl, conduct an experiment that became known as the methylsen and style experiment. And what this showed was that the semiconserved replication hypothesis correctly described the way that DNA molecules actually replicated. Now, before we discuss what the experiment looked like, let's actually take a look at what this hypothesis tells us. So based on the semiconserve replication of DNA we have our DNA molecule the original DNA molecule that consists of this double helix that contains two individual strands of DNA that we're going to call the parent strands or the parental strands so we have parental strand number one and it's complementary parental strand number two shown in blue. Now, before replication begins, we have to separate our DNA molecule. And so we separate the two individual strands of DNA and once we separate them, each one of these blue strands, the original parenthal strands, act as a template to synthesize the complementary new strand we're going to call the daughter strand that is shown in green. | Meselson and Stahl Experiment .txt |
So based on the semiconserve replication of DNA we have our DNA molecule the original DNA molecule that consists of this double helix that contains two individual strands of DNA that we're going to call the parent strands or the parental strands so we have parental strand number one and it's complementary parental strand number two shown in blue. Now, before replication begins, we have to separate our DNA molecule. And so we separate the two individual strands of DNA and once we separate them, each one of these blue strands, the original parenthal strands, act as a template to synthesize the complementary new strand we're going to call the daughter strand that is shown in green. And following the replication process, this will be the distribution, the arrangement of the parent strand and the daughter strand. So in each one of these two DNA molecules, one of these strands will be conserved, it will be that original parental strand and the other one will be the complementary newly synthesized daughter strand. And so we have this semiconservative process in which we have 50% of that original parental strand and 50% of that newly synthesized daughter strand. | Meselson and Stahl Experiment .txt |
And following the replication process, this will be the distribution, the arrangement of the parent strand and the daughter strand. So in each one of these two DNA molecules, one of these strands will be conserved, it will be that original parental strand and the other one will be the complementary newly synthesized daughter strand. And so we have this semiconservative process in which we have 50% of that original parental strand and 50% of that newly synthesized daughter strand. And these two DNA molecules are essentially identical, assuming that process was mitosis. Now, let's actually discuss what this experiment consisted and how it showed that the semiconservative replication process was correct. So what they did initially was they wanted to grow these special bacterial E. Coli cells that contain labeled DNA molecules. | Meselson and Stahl Experiment .txt |
And these two DNA molecules are essentially identical, assuming that process was mitosis. Now, let's actually discuss what this experiment consisted and how it showed that the semiconservative replication process was correct. So what they did initially was they wanted to grow these special bacterial E. Coli cells that contain labeled DNA molecules. So radioactively labeled DNA molecules. And the way that they did that is they took a medium that was very high, very rich in isotopic nitrogen atoms. So m 15. | Meselson and Stahl Experiment .txt |
So radioactively labeled DNA molecules. And the way that they did that is they took a medium that was very high, very rich in isotopic nitrogen atoms. So m 15. Now, remember, the normal nitrogen atom contains an atomic mass of 14, and that's because it contains seven protons and seven neutrons in the nucleus. But in the case of this heavy isotopic nitrogen atom, we have eight neutrons and seven protons. And so eight plus seven gives us an atomic mass of 15. | Meselson and Stahl Experiment .txt |
Now, remember, the normal nitrogen atom contains an atomic mass of 14, and that's because it contains seven protons and seven neutrons in the nucleus. But in the case of this heavy isotopic nitrogen atom, we have eight neutrons and seven protons. And so eight plus seven gives us an atomic mass of 15. So initially, E. Coli bacterial cells were grown in a medium rich in heavy nitrogen and 15, as shown in the following diagram. And this was done to basically produce E. Coli cells that have incorporated this isotopic N 15 into their DNA. And now the DNA is radioactively labeled and contains the heavier nitrogen atoms. | Meselson and Stahl Experiment .txt |
So initially, E. Coli bacterial cells were grown in a medium rich in heavy nitrogen and 15, as shown in the following diagram. And this was done to basically produce E. Coli cells that have incorporated this isotopic N 15 into their DNA. And now the DNA is radioactively labeled and contains the heavier nitrogen atoms. Now once we form these E. Coli cells that contain the heavy DNA molecules, these ecology cells are then transferred into a beaker into a flask that contains the regular nitrogen N 14 isotope. And so now when these E. Coli cells are going to reproduce via mitosis, they're going to incorporate the N 14 isotope into the newly synthesized DNA molecules. And what we want to answer is what will be the distribution of the N 14 and N 15 isotopes following several cycles of replication. | Meselson and Stahl Experiment .txt |
Now once we form these E. Coli cells that contain the heavy DNA molecules, these ecology cells are then transferred into a beaker into a flask that contains the regular nitrogen N 14 isotope. And so now when these E. Coli cells are going to reproduce via mitosis, they're going to incorporate the N 14 isotope into the newly synthesized DNA molecules. And what we want to answer is what will be the distribution of the N 14 and N 15 isotopes following several cycles of replication. And based on this, we can basically determine if the semiconservative process actually works. So this experiment has three important points. Number one is the original parental strands contain the N 15 isotopes and that's because they were initially grown in the N 15 rich medium b is or .2 is replicated daughter strands because now the medium is changed to the N 14. | Meselson and Stahl Experiment .txt |
And based on this, we can basically determine if the semiconservative process actually works. So this experiment has three important points. Number one is the original parental strands contain the N 15 isotopes and that's because they were initially grown in the N 15 rich medium b is or .2 is replicated daughter strands because now the medium is changed to the N 14. The replicated daughter strands will contain N 14 isotopes because as these daughter cells are synthesized we're going to take the nitrogen to synthesize the nitrogenous bases and sugars and so forth from this median that contains the M 14. So the newly synthesized strands will contain the regular N 14 isotope. But the other ones, the original will contain that labeled N 15 isotope. | Meselson and Stahl Experiment .txt |
The replicated daughter strands will contain N 14 isotopes because as these daughter cells are synthesized we're going to take the nitrogen to synthesize the nitrogenous bases and sugars and so forth from this median that contains the M 14. So the newly synthesized strands will contain the regular N 14 isotope. But the other ones, the original will contain that labeled N 15 isotope. Now, why did we want to use N 15 and N 14? Well, because they have a difference in mass the DNA molecules, the old ones and the new ones will also different mass and we can separate them via the process of centrifugation which separates bimass by size and by density. So the DNA with different densities can be separated by centrifugation. | Meselson and Stahl Experiment .txt |
Now, why did we want to use N 15 and N 14? Well, because they have a difference in mass the DNA molecules, the old ones and the new ones will also different mass and we can separate them via the process of centrifugation which separates bimass by size and by density. So the DNA with different densities can be separated by centrifugation. So what we basically do is we allow these cells, the equalized cells, to replicate, to divide and replicate and we extract the DNA. Then we centrifuge that DNA and we analyze our DNA. So what they obtained was the following three photographs. | Meselson and Stahl Experiment .txt |
So what we basically do is we allow these cells, the equalized cells, to replicate, to divide and replicate and we extract the DNA. Then we centrifuge that DNA and we analyze our DNA. So what they obtained was the following three photographs. So each of these photographs basically describes the band that correlates to the DNA molecules that were extracted. So if we take out our DNA molecule from our equalized cell that has not yet divided, then what we'll see is a single band that describes a single type of DNA molecule. Now, by the way, as we go this way from left to right along the x axis, we increase in density. | Meselson and Stahl Experiment .txt |
So each of these photographs basically describes the band that correlates to the DNA molecules that were extracted. So if we take out our DNA molecule from our equalized cell that has not yet divided, then what we'll see is a single band that describes a single type of DNA molecule. Now, by the way, as we go this way from left to right along the x axis, we increase in density. And so if this is our test tube, this way it's towards the bottom of that test tube and this way it's towards the top of that test tube. And so if the band appears lower that means the DNA molecule that is described by that band has a higher density because the farther we go to the right along the x axis, the more dense our molecule is. And so initially what our initial sample showed was we had a single band. | Meselson and Stahl Experiment .txt |
And so if this is our test tube, this way it's towards the bottom of that test tube and this way it's towards the top of that test tube. And so if the band appears lower that means the DNA molecule that is described by that band has a higher density because the farther we go to the right along the x axis, the more dense our molecule is. And so initially what our initial sample showed was we had a single band. Now this single band basically described a single type of DNA molecule in which both of those strands consisted of that isotopic N 15 atom. So this describes the N 15 DNA molecule and so this is what we see. So one of these strands consists of the N 15 and the other strand also consists of the N 15. | Meselson and Stahl Experiment .txt |
Now this single band basically described a single type of DNA molecule in which both of those strands consisted of that isotopic N 15 atom. So this describes the N 15 DNA molecule and so this is what we see. So one of these strands consists of the N 15 and the other strand also consists of the N 15. And that's because the initial equalize cells that we took out from this beaker consisted of these radioactively labeled DNA molecules. So before replication occurred, all cells had heavy DNA and this was represented by a single band along the following diagram. Now, following one replication, when we extract the DNA from the first generation cells that came from those initial equalized cells, this is the band that we found. | Meselson and Stahl Experiment .txt |
And that's because the initial equalize cells that we took out from this beaker consisted of these radioactively labeled DNA molecules. So before replication occurred, all cells had heavy DNA and this was represented by a single band along the following diagram. Now, following one replication, when we extract the DNA from the first generation cells that came from those initial equalized cells, this is the band that we found. And notice we also had a single band just like in this case. But this band was shifted to the left along this diagram, along the photograph. And what this basically means is we have a single type of DNA molecule because we have that single band but it is lighter, less dense than in this particular case because it is found farther to the left along the x axis. | Meselson and Stahl Experiment .txt |
And notice we also had a single band just like in this case. But this band was shifted to the left along this diagram, along the photograph. And what this basically means is we have a single type of DNA molecule because we have that single band but it is lighter, less dense than in this particular case because it is found farther to the left along the x axis. And that essentially confirmed the fact that after one generation, after one cycle we form one type of DNA molecule that consists of one radioactively labeled strand and one newly synthesized strand that contains the lighter N 14 isotopes. And so this is what we saw. So one of these was N 15 and the other one was N 14 because it was synthesized using the nitrogen atoms that had an atomic mass of 14 and not 15. | Meselson and Stahl Experiment .txt |
And that essentially confirmed the fact that after one generation, after one cycle we form one type of DNA molecule that consists of one radioactively labeled strand and one newly synthesized strand that contains the lighter N 14 isotopes. And so this is what we saw. So one of these was N 15 and the other one was N 14 because it was synthesized using the nitrogen atoms that had an atomic mass of 14 and not 15. And because of their lighter nature they're going to be less dense and so they're going to appear farther to the left along that x axis. Now, what do they see after a second cycle of division? So following a second division, a second replication, once we extract the DNA molecules from those cells, this is what we saw. | Meselson and Stahl Experiment .txt |