Denaturation, Tests and Calculations

Transcript

0:00Introduction

Hello everyone! Crack an egg, and the egg white is clear and runny. Boil it for a few minutes, and the same egg white becomes a firm white solid. Now here is the puzzle. Not a single peptide bond of the protein chain has been broken, and the sequence of amino acids is exactly the same. So what has changed? Keep that question in mind. Today we look at denaturation, four colour tests for proteins, and a neat calculation that counts the iron atoms in haemoglobin from one percentage and the molar mass. Questions on these do come up in JEE chemistry, so let's begin.

0:40Denaturation: the shape is lost

So, on the right are the four levels of protein structure, from left to right: primary, secondary, tertiary and quaternary. Now, a protein in its natural, biologically active shape is called a native protein. Okay? This shape is delicate. Heating, or a change of p H, disturbs the H-bonds, short for hydrogen bonds, that hold the shape together. Right? Then the helices uncoil, and the globules unfold. With its shape gone, the protein loses its biological activity, and this change is called denaturation. With me? Now let me ask you this. Look at the figure. When a protein is denatured, which levels of structure are destroyed, and which one survives? Yes, exactly, the secondary and tertiary structures are lost, and the quaternary structure too, as the brackets say, when the protein has one. So the alpha helix and the beta sheet, marked with the Greek letters alpha and beta, and the overall folding in space all go. Clear? But the primary structure remains intact. The chain of amino acids on the left, joined by peptide bonds, is not broken at all. So here is the answer to our egg puzzle: the sequence stays exactly the same, and only the shape is lost. Got it?

1:56Denaturation in everyday life

The course gives two everyday examples. First, our egg: egg white coagulates on boiling, so that is heat at work. The second is curd. When bacteria grow in milk, it curdles without any heating. So, of our two causes, which one is at work here? It is the change of p H, because the bacteria produce lactic acid, and the acid curdles the milk. So, heat for the egg, and acid for the milk. Okay?

2:25Tests for proteins: biuret and xanthoproteic

Now, the course gives four colour tests for proteins. The first is the biuret test. We add N a O H, sodium hydroxide, and a little C u S O four, copper sulphate; the small four is a subscript. The colour that appears is violet. Right? And this violet colour is given by compounds with two or more peptide bonds. Hold on to those words, two or more. Clear? So tell me, a dipeptide is two amino acids joined together. Does it give the biuret test? No, it does not, and that is the trap. Two amino acids are joined by only one peptide bond, and the test needs at least two. So a tripeptide, with two peptide bonds, does give the violet colour. With me? The second is the xanthoproteic test. We add conc, short for concentrated, HNO three, which is nitric acid. The colour is yellow, and it turns orange with alkali. Okay? This one is given by proteins with aromatic side chains. So it tells you about the side chains, not about the peptide bonds. Got it?

3:32Tests for proteins: Millon and ninhydrin

The third is Millon's test. The reagent is mercury one and mercury two nitrate in nitric acid; the Roman numerals in brackets are the oxidation states of mercury. The colour this time is red. Right? It is given by proteins containing tyrosine, which, as the brackets say, is a phenol. Clear? And the fourth is the ninhydrin test. Warm the solution with ninhydrin, and a purple colour appears. Okay? This one is given by amino acids and by proteins. So even a free amino acid turns purple here, unlike in the biuret test. With me? Here is a quick check for you. Suppose a solution gives a purple colour with ninhydrin, but no colour in the biuret test. Does it contain a protein? No, it does not, because a protein would have turned the biuret test violet. So the solution has amino acids, or at most a dipeptide, and no protein. Got it?

4:29Minimum molar mass from a percentage

Now, the calculation part. Suppose a protein contains p percent by mass of some element, or of some residue; that is the p with the percent sign. And m is the molar mass of that element or residue. Okay? Every molecule must contain at least one of it, since a molecule cannot hold a fraction of an atom, or of a residue. Right? So take the smallest case, just one per molecule. Then in one mole of the protein, m grams is p percent of the molar mass. That gives M min, capital M with a small min written below, the minimum molar mass, equal to a hundred times m, over p. Clear? It is only a minimum, because a molecule may contain more than one. If it contains n of them, the actual molar mass, capital M, is n times M min. With me?

5:18Example 1: minimum molar mass from 0.80% sulphur

Time for the first example. The question says that a protein contains zero point eight zero percent of sulphur. Find its minimum molar mass. In the brackets, S equals thirty two gives the molar mass of sulphur. Pause the video and try it first. Done? Okay, let's check. Here m is thirty two, and p is zero point eight zero. So M min is a hundred times thirty two, over zero point eight zero. Notice that p goes in as the percentage itself, not as a fraction, because the hundred is already in the formula. Right? One hundred times thirty two is three thousand two hundred, and three thousand two hundred over zero point eight zero gives four thousand. So the minimum molar mass is four thousand grams per mole; the g, mol to the power minus one on screen is read as grams per mole.

6:13Example 2: iron atoms in haemoglobin from 0.34% iron

Clear? Now a harder one, the haemoglobin calculation from the start. Haemoglobin contains zero point three four percent of iron by mass. Find its minimum molar mass. Its actual molar mass is about sixty five thousand nine hundred. How many iron atoms does one molecule contain? In the brackets, F e, the symbol for iron, has molar mass fifty six. Pause the video and try it first. Done? Okay, let's check. Here m is fifty six, and p is zero point three four. Fifty six times a hundred is five thousand six hundred, and five thousand six hundred over zero point three four gives about sixteen thousand four hundred and seventy. The wavy equals sign means approximately, so that is about one point six five times ten to the power four grams per mole. Okay? So the molar mass of haemoglobin is about sixteen thousand four hundred and seventy, and we are done, right? Careful, because that is the trap. That value is only the minimum, for one iron atom per molecule. The actual molar mass is about sixty five thousand nine hundred, so n, the number of iron atoms, is sixty five thousand nine hundred over sixteen thousand four hundred and seventy, which is about four. Clear? So one molecule of haemoglobin contains four iron atoms. From one percentage and the actual molar mass, we have counted the iron atoms in a giant protein. See how neat that was?

7:42Quick revision: denaturation, four tests, minimum molar mass

So, quick revision. In denaturation, the hydrogen bonds are disturbed, the shape and the biological activity are lost, but the primary structure stays intact. The four tests: biuret gives violet, xanthoproteic yellow, Millon red, and ninhydrin purple. And the minimum molar mass is a hundred times m, over p, while the actual molar mass is n times M min. Now go and try the practice problems on this page. All the best!