A student was given a task of identifying the contents of five bottles of amino acids from which the labels had fallen off. Each of the original bottles contained one of the following: arginine, histidine, cysteine, proline, and tryptophan.Show answer
162. Which of the following methods could be most
readily employed to identify tryptophan?
(A) Electrophoresis
(B) Ultraviolet spectroscopy
(C) Gel filtration
(D) Analytical ultracentrifugation
(E) Optical rotation
163. Each amino acid was subjected to paper electrophoresis in a pH 9.5 buffer, and then the amino acids were visualized by spraying the paper with ninhydrin. Which amino acid is at the point labeled (1) in the figure above?
(A) Arginine
(B) Histidine
(C) Cysteine
(D) Proline
(E) Tryptophan
First one question is rather easy. Tryptophan have one very useful feature that called fluorescence. That is emission of light after illumination. Trp is one of 20 amino acids and widely occur in proteins and its fluorescence provide useful data about protein's folding and structure. Trp's fluorescence is one of the most intense among all fluorescent amino acids (others are Phe and Tyr). Trp excitation wave length is 280 nm and emitted light has wave length 300-350 nm. Ultraviolet is part of spectrum from 10 to 400 nm. So right answer is B, because other amino acids from the list are not fluorescent.
Next part of question refers mainly to amino acids' behavior in different pH. So it is necessary to investigate polarity of substances in solutions with different acidity.
But what does pH actually means? It is defined as pH=-Log[H+], i.e. minus decimal logarithm of H+ (protons) concentration in solution. Neutral pH is 7 because in water there are 10^-7 M/liter of H+ ions due to H20 decay into H+ and OH-.
So, briefly, high pH means very low concentration of H+, and low pH's mean high concentration of H+, or low concentration of OH-. Actually, pH and pOH (defined in the same manner) are closely connected with each other. For pure water pH=pOH, of course.
Now lets go further. Practically all amino acids can be distinguished using pKa – pH level at which molecule is not charged. That very simple experiment described in the question. If there is any pKa and if acid (actually, amino acid residue) have NH2 or OH groups, then at pH higher than pKa protons will be taken away from acid so -OH will become -O- (oxygen with negative charge) and at lower that pKa pH acid's NH2 will become NH3+ (one positive charge). Alberts provide several examples, that make everything clear:
Using pKa values is quite hard to solve this problem, really. Best picture can be granted by pI value. That is isoelectric point, pH level, at which whole molecule of acid is electroneutral. There is a nice table of pI values on the web. If pH higher than pI, then protons will be taken away and charge will become negative. Otherwise at pH lower than pI molecule will be protonated. If you check linked page, you will find, that all molecules from questions have pI lower than 9.5 and only arginine have higher value. Hence, Pro, His, Cys and Trp will have negative charge and only Arg – positive. That is why four molecules will move on one side from point of sample application, and only one – on the other. So it will be Arg, the answer is A. Note, that picture is electrophoresis results, so + and - are signs of CAthode and ANode, which attracts CAtions and ANions respectively.
Additional information
OK, we figured out – using something called pI – right answer. But why does it happens to be right? And why – what is more important – why argynine differs so much from other amino acids?
First, what is pI actually? It is mean of pK(amino group) and pK(carboxyl group) for molecule, which have only one of those. pI=(pK(NH2)+pK(-OH))/2. When pH is rising from pK of -OH more and more groups undergo deprotonating. Similarly, when pH decrease from pK of NH2, more and more groups undergo protonating, so there is point of equilibrium, where number of protonated groups equal number of deprotonated groups, thus net charge is zero. We assume that this equilibrium point is average of pK's.
But there are several amino acid residues that have their own -NH2 or -OH groups and even not one as Arg (2 -NH2), His (1) and so on. In this case pI is counted as sum of pK-s of all -OH and -NH2 groups divided by two. That is why Arg has pI much greater than any amino acid's that lack -NH2 groups at all.
Next part of question refers mainly to amino acids' behavior in different pH. So it is necessary to investigate polarity of substances in solutions with different acidity.
But what does pH actually means? It is defined as pH=-Log[H+], i.e. minus decimal logarithm of H+ (protons) concentration in solution. Neutral pH is 7 because in water there are 10^-7 M/liter of H+ ions due to H20 decay into H+ and OH-.
So, briefly, high pH means very low concentration of H+, and low pH's mean high concentration of H+, or low concentration of OH-. Actually, pH and pOH (defined in the same manner) are closely connected with each other. For pure water pH=pOH, of course.
Now lets go further. Practically all amino acids can be distinguished using pKa – pH level at which molecule is not charged. That very simple experiment described in the question. If there is any pKa and if acid (actually, amino acid residue) have NH2 or OH groups, then at pH higher than pKa protons will be taken away from acid so -OH will become -O- (oxygen with negative charge) and at lower that pKa pH acid's NH2 will become NH3+ (one positive charge). Alberts provide several examples, that make everything clear:
Using pKa values is quite hard to solve this problem, really. Best picture can be granted by pI value. That is isoelectric point, pH level, at which whole molecule of acid is electroneutral. There is a nice table of pI values on the web. If pH higher than pI, then protons will be taken away and charge will become negative. Otherwise at pH lower than pI molecule will be protonated. If you check linked page, you will find, that all molecules from questions have pI lower than 9.5 and only arginine have higher value. Hence, Pro, His, Cys and Trp will have negative charge and only Arg – positive. That is why four molecules will move on one side from point of sample application, and only one – on the other. So it will be Arg, the answer is A. Note, that picture is electrophoresis results, so + and - are signs of CAthode and ANode, which attracts CAtions and ANions respectively.
Additional information
OK, we figured out – using something called pI – right answer. But why does it happens to be right? And why – what is more important – why argynine differs so much from other amino acids?
First, what is pI actually? It is mean of pK(amino group) and pK(carboxyl group) for molecule, which have only one of those. pI=(pK(NH2)+pK(-OH))/2. When pH is rising from pK of -OH more and more groups undergo deprotonating. Similarly, when pH decrease from pK of NH2, more and more groups undergo protonating, so there is point of equilibrium, where number of protonated groups equal number of deprotonated groups, thus net charge is zero. We assume that this equilibrium point is average of pK's.
But there are several amino acid residues that have their own -NH2 or -OH groups and even not one as Arg (2 -NH2), His (1) and so on. In this case pI is counted as sum of pK-s of all -OH and -NH2 groups divided by two. That is why Arg has pI much greater than any amino acid's that lack -NH2 groups at all.
