4. Amino acids, peptides and proteins

Question 45. What compounds belong to a-amino acids? What is a-amino acid role in living organism functioning?

Answer. a-Amino acids are hetero-functional compounds which contain two functional groups: carboxylic group (–СООН) and amino group (–NH₂), both groups are attached to one and the same carbon atom. a-Amino acids are named according to International nomenclature, and carbon atom with amino group is the second carbon atom. Many a-amino acids, especially those in the protein structure, are used in their trivial names according to trivial nomenclature.

N umerous peptides and proteins at living organisms are built from twenty a-amino acid residues. All a-amino acids contain one and the same fragment, H₂N–СН–СООН group, second carbon atom is attached to hydrocarbon radical –R. Hydrocarbon radical could have different chemical nature.

Modern rational amino acid classification is based on radical polar character, i.e. their ability to react with water at physiological pH (which are close to pH 7,0). Amino acids could have the following radicals: 1) non-polar (hydrophobic); 2) polar (hydrophilic); 3) aromatic; 4) negatively charged and 5) positively charged. There are amino acid structural formulas, their names according to trivial nomenclature, abbreviated English names and one-letter symbols which are accepted in foreign and Russian literature.

a-Amino acids with non-polar (hydrophobic) radical

a-Amino acids with polar un-charged radical

a-Amino acids with polar negatively charged radical

a-Amino acids with polar positively charged radical

a-Amino acids with aromatic radical

a-Amino acids are colourless crystals, soluble in water under normal conditions.

Protein synthesis in the organism is possible only all twenty a-amino acids are presented in the organism. Main source of a-amino acids is food proteins. Some a-amino acids could be synthesized in the organism (from other amino acids or other metabolic products), but some part of a-amino acids is not synthesized at human organism and could come only with food, that is why balanced protein nutrition is very important for human beings. Such a-amino acids are called essential: valine, leucine, isoleucine, lysine, threonine, methionine, phenylalanine, tryptophan.

At some diseases non-essential amino acids could become essential. For example, at phenylketonuria organism stops synthesizing tyrosine.

Many a-amino acids are used at Medicine. For example, glycine is used to make metabolic process in brain tissues and muscles better. Glutamic acid is used as a treatment of central nervous system dis-orders, methionine and histidine are used for cure hepatic diseases etc.

Question 46. Write formulas of stereo-isomers (enantiomers) for a-amino acids. Are all natural a-amino acids optically active?

Answer. At all natural a-amino acid molecules, except for glycine, one and the same carbon atom (the second) is attached to four different substitutes: carboxylic group (–СООН), amino group (–NH₂), hydrocarbon radical R and hydrogen atom, i.e. at least one carbon atom from all carbon atoms in these compounds is asymmetric (chiral). At all natural a-amino acids there are no elements of symmetry, thus, all a-amino acids (except glycine) are optically active compounds and could exist in the form of two enantiomers. To write enantiomers properly we should locate carbon chain vertically. Group of the highest priority (carboxylic) is located at the top. Two other substitutes (amino group and hydrogen atom) at second (asymmetric) carbon atom are located at the right and at the left in relation to carbon chain. In standard Fischer’s formula L-configuration corresponds to amino group location at the left from the carbon chain, in D- configuration amino group is located in the right:

Amino acids isoleucine and threonine have two asymmetric carbon atoms (second and third) in the molecule, thus, they could exist in the form of two enantiomer pairs:

From all four isomers only one is used in human organism to build proteins: L-threonine and L-isoleucine.

Other a-amino acids in the protein structure are in L-form. a-Amino acids of D-form are in peptide structure at some micro-organisms and bacteria.

Question 47. What is acid-base property expression and amphoteric character at a-amino acids? What a-amino acid properties determine their ion structure?

Answer. a-Amino acids form salts with both alkali and acids as they are amphoteric compounds, as in their molecules there are acid (-COOH) and base (-NH₂) functional groups:

W ith heavy metal cations a-amino acids form inner-salts of chelate (claw-like) type:

Arrow directed from nitrogen atom to copper atom in chelate formula stands for chemical bond formed according to donor-acceptor mechanism. Nitrogen atom donates its electron pair to form a bond, it is a donor, and copper atom providing vacant (free) orbital is an acceptor.

A t aqueous solutions amphoteric a-amino acids exist in the form of balanced mixture of di-polar ion, cation and anion forms:

At strong acid solutions (рН = 1-2) cation form predominates, at strong alkaline solutions (рН = 13-14) anion form predominates. When solid a-amino acids exist in the form of di-polar ions. At equation of reactions with a-amino acids they are written in non-ionized form.

Ionic structure of a-amino acid molecules determines their high melting temperatures (higher than 200 °С), non-volatility, water-solubility, non-solubility at organic solvents. Solubility in water provides a-amino acid biological functions: absorption, transmission in the organism etc.

Question 48. What is the medium reaction at a-amino acid dissolving at distilled water?

Answer. Solution рН, received at a-amino acid dissolving depends on functional group nature at acid hydrocarbon radical, to be more particular it depends on number of acid and base groups.

Let’s analyze amino acids with non-polar hydrocarbon radical. In the molecule there is one acid group (–СООН) and one basic group (–NH₂). In aqueous solutions these amino acids are ionized. Distilled water used for solutions does not have excessive amount of Н⁺ ions, and ОН^– ions, medium is neutral (рН = 7,0), thus, ion forms in the solution are formed due to Н⁺ ions transition from acid group (–СООН) to basic group (–NH₂). Practically all amino acid molecules exist in the form of di-polar ions and medium could be considered close to neutral.

At glutamic and aspartic acid molecules there are two acid carboxylic groups (–СООН) and one basic group (–NH₂). At solution there is also Н⁺ ion transition from carboxylic group to amino group in a-position. Proton formed at second carboxylic group dissociation transits to solution. Thus, excessive amount of Н⁺ ions is accumulated in the solution, medium is acid (рН < 7).

Amino acids lysine, arginine and histidine have one carboxylic group and two basic amino groups. In the solution there is Н⁺ ion transition from carboxylic group to amino group at a-position. At second amino group interaction with water in the solution there is excessive amount of ОН^– accumulation, so medium is alkaline (рН > 7).

Question 49. What is a-amino acid isoelectric point? In what way a-amino acids move in the electric field?

Answer. Medium рН, at which practically all amino acid molecules exist in the form of bi-polar ions, is called amino acid isoelectric point for this amino acid and is marked as рI. At isoelectric point total charge on a-amino acid molecule is zero. Di-polar ions do not move in the electric field, thus, at medium рН which is рI, amino acid solution does not conduct electric current.

If medium рН is lower than рI, amino acid exists predominantly as cation. Under the action of constant electric field, for example, at electrophoresis, amino acid cation moves to negatively charged electrode (cathode).

If medium рН is higher than рI, amino acid predominantly exists in the form of anion. Under the action of constant electric field, for example, at electrophoresis, amino acid anion moves to positively charged electrode (anode).

At acid medium a-amino acid is cation, moves to cathode and is reduced there. At basic medium (alkaline) a-amino acid is anion, moves to anode and is oxidized there.

With the help of electrophoresis it is possible to separate individual amino acids at a-amino acid mixture.

For example, there is mixture of three acids: alanine (рI = 6,1), lysine (рI = 9,7) and glutamic acid (рI = 3,2). Let’s try to define their movement direction if electrophoresis is performed at рН = 6,1.

For lysine medium electrophoresis рН is lower than рI (6,1 < 9,7), thus, in the solution lysine predominantly exists in the form of cation and under the action of electric field it moves to cathode.

For glutamic acid medium electrophoresis рН is higher than рI (6,1 > 3,2), Thus, in the solution glutamic acid predominantly exists in the form of anion and under the action of constant electric field it moves to anode. As alanine isoelectric point is 6,1 then it will stay at the start line (it will not move anywhere).

Question 50. Write down valine structural formula. Name this amino acid according to international nomenclature. Write down isomers on position of functional group, carbon chain structure, functional group. Is valine optically active? If it is optically active write and name its enantiomers. What is medium pH of this amino acid solution at distilled water? What is ion form of this amino acid at gastric juice (рН = 1)? What is medium pH when this amino acid solution does not conduct electric current?

Answer. To give valine a name according to international nomenclature we should find the longest carbon chain, enumerate it starting from functional group with the highest priority (carboxylic group). In the chain there are four carbon atoms then it is butanoic acid, which has amino group at the second carbon atom and which has methyl group at the third carbon atom. Carbon chain is branched, to write isomer on the structure of the carbon chain it is enough to locate all carbon atoms as a line and we get an isomer: 2-amino-pentanoic acid. To write an isomer on position of the functional group it is possible to transmit amino group to the third carbon atom and transmit methyl group to the second carbon atom. We receive 3-amino-2-methyl-butanoic acid. To write the third isomer it is necessary to change some functional group, for example, at changing –ОН group on to amino group we receive 2-hydroxy-3-methyl-butanoic acid amide.

Valine is optically active as in its structure there is asymmetric (chiral) carbon atom (С₂) and there are no symmetry elements, thus, this compound could be represented as two absolute configurations (R- and S-isomers), and in the form of two relative configurations (L- and D-isomers).

In molecule structure there is one carboxylic group and one amino group, valine is amphoteric compound, and its solution in distilled water has neutral medium. At strong acid medium (рН = 1) valine is protoned on amino group and it is cation. рН, at which amino acid solution does not conduct electric current is called isoelectric point; for valine it is 6,0.

Question 51. What reaction could transform isoleucine into 3-methyl-2-pentenoic acid? What reaction could transform serine into 2-amino-ethanol?

Answer. Let’s write down formulas of initial compound and first reaction product.

It is obvious that to receive 3-methyl-2-pentenoic acid we need removing amino group from isoleucine. If we remove amino group then at second carbon atom there is one bond released:

To form double bond between the second and third carbon atoms we need one free bond at the third carbon atom. Methyl group in this situation (–СН₃) should be preserved at a molecule. It could be done eliminating hydrogen atom from the third carbon atom:

СН₃–СН₂–СН–СН–С ® СН₃–СН₂–С=СН–С

ОН

О

СН₃

–Н

ОН

О

СН₃

1

2

3

4

5

1

2

3

4

5

Thus, to receive 3-methyl-2-pentenoic acid isoleucine molecule should lose amino group and hydrogen atom. In free form these particles are not stable and form ammonia molecule NН₃. If ammonia molecule is discharged from amino acid molecule with unsaturated acid formation, then these are the signs of intra-molecular deamination reaction (question 36). Final equation of reaction could be written in the following way:

Now let’s write down serine and 2-amino-ethanol molecules:

Comparing formulas we come to decision that to solve our problem we need removing carbon atom and two oxygen atoms from carboxylic group. During reaction serine should lose carboxylic group. This type of reaction is called decarboxylation, carboxylic group is discharged in the form of carbon tetra-oxide.

In the organism a-amino acid decarboxylation goes on under enzyme action which is called decarboxylase. Equation of reaction could be written in the following way:

Question 52. What compound is formed at serine reductive deamination and glutamic acid oxidative deamination? Do oxidative deamination products differ from each other in vivo (in the organism) and in vitro (in the test tube)?

Answer. At a-amino acid reductive deamination hydrogen atom in the second position is substituted. One more hydrogen atom adds amino group with ammonia molecule formation:

As a result 3-hydroxy-propanoic acid is formed.

a-Amino acid oxidative deamination in living organism has two stages. At the first stage under the action of enzymes dehydrogenases there is dehydrogenation (hydrogen elimination) of initial a-amino acid and imino acid is formed. At the second stage due to imino acid hydrolysis (interaction with water) there is a-keto acid and ammonia formation. For glutamic acid these stages have the following form:

At laboratory conditions oxidative deamination is performed with the help of nitrous acid. There is a-hydroxy acid formation and gas nitrogen discharge:

Question 53. What compounds are formed in the organism at interaction between valine and pyruvic acid?

A nswer. Let’s write down initial compound formulas:

Valine is a-amino acid, pyruvic acid is a-keto acid. In the living organism under the action of enzymes which are transaminases, there is transamination reaction, i.e. amino group transition. Amino group donor is a-amino acid and its acceptor is a-keto acid. a-Amino acid is transformed into a-keto acid and a-keto acid due to reaction is transformed into a-amino acid with corresponding radical structure (amino group and keto group exchange their positions).

At interaction between valine and pyruvic acid there is the following reaction:

Reaction products are 2-oxo-3-methyl-butanoic acid and 2-amino-propanoic acid (alanine). This reaction is used in the organism to synthesize non-essential amino acids and to remove excessive amount of un-necessary amino acids and connection is formed between protein and carbohydrate metabolism.

Question 54. What are peptides? What is their difference from proteins? What reaction forms peptide in the organism?

Answer. Peptides and proteins are compounds built from a-amino acid residues. It is considered that peptides could have up to 100 amino acid residues and proteins could have more than 100 amino acid residues. Peptides are subdivided into oligopeptides (low-molecular peptides) which contain less than 10 amino acid residues. Peptides containing higher number of amino acid residues are called poly-peptides. For macro-molecules with amino acid residues number close to 100 or excessing 100, term peptide practically corresponds to protein. Poly-peptide and protein chains are formed as a result of a-amino acid poly-condensation reaction with peptide (amide) bond formation (–СО–NH–) between monomer parts:

Poly-peptide (poly-amide) chains at peptides and proteins, as a rule, have unbranched structure. Terminal of the chain with amino acid with free amino group is called N-terminal of the peptide (protein), the other terminal with amino acid with free carboxylic group is called C-terminal of peptide (protein).Peptide and protein chains are written starting with N-terminal. Peptides are in all living organisms. In opposition to proteins they have more varied amino acid content which sometimes includes D-amino acids. They could have cyclic fragments, branched chains, etc. Peptides having high physiologic activity regulate different biological processes. According to bio-regulatory action peptides are subdivided into several groups:

– compounds characterized by hormonal activity (glucagon, oxytocine, vasopressine, etc.);

– compounds regulating digestive process (gastrin, gastric inhibiting peptide, etc.);

– peptides regulating appetite (endorphins, neuropeptide-Y, leptin, etc.);

– compounds having pain relieving effect (opioid peptides);

– substances regulating higher nervous activity, biochemical processes, connected to memory, education processes, fear, anger appearance etc.;

– peptides regulating arterial pressure and vascular tone (angiotensin II, bradicardine, etc.).

This subdivision is conditional as various peptides action is not limited by only one direction. For example, vasopressine in addition to vaso-constricting and anti-diuretic action also makes the memory better.

All enzymes have protein nature.

Question 55. Write down tri-peptide formula which condensed marking is ala-lys-asp? What is this tri-peptide name? What is the process of enzymatic hydrolysis under the enzyme trypsin which splits only peptide bonds formed by carboxylic group of lysine or arginine?

Answer. Amino acids have markings containing three letters which are first three letters in their trivial name. Аla stands for alanine, lys stands for lysine, asp stands for aspartic acid. According to the wording of the task N-terminal is amino group from alanine, C-terminal is carboxylic group from aspartic acid. To write peptide properly all three amino acids should be located near each other so that on the one line there is amino group, a-carbon atom and carboxylic group. Amino acid radicals are located vertically (up or down). Carboxylic group of one amino acid takes part at peptide bond formation (–ОН group is discharged from it) and a-amino group of the second amino acid (hydrogen atom is discharged from it). Carboxylic and amino groups which are in hydrocarbon radical do not take part at peptide bond formation.

Peptide names are made due to successive enumeration of amino acid residues starting from N-terminal of the chain. And ending “-ine” at the trivial acid name is changed into “-yl”, exception is amino acid which is C-terminal (its name is not changed). In our case tri-peptide is called alanyl-lysyl-aspartic acid.

As trypsin splits only peptide bonds formed by lysine carboxylic group (there is no arginine at or peptide) then equation of enzymatic hydrolysis has the following form:

Question 56. Characterize primary, secondary, tertiary and quaternary protein structure.

Answer. Primary protein structure is succession of alterations between a-amino acids at poly-peptide chain (figure 1):

Figure 1. Insulin primary structure.

Amino acids are attached to each other by peptide bonds. Primary structure can be identified due to successive terminal a-amino acid splitting and their defining.

Secondary structure is represented by two conformations (fig.2). One structure from secondary protein structures is helix (this structure is also called a-structure or a-helix). This poly-peptide chain conformation is right-wind spiral. It is possible to imagine it as poly-peptide chains winds around cylinder surface clock-wise (to the right). One round of spiral contain average number of 3,6 amino acid residues (36 residues at 10 rounds); spiral step (distance between two neighboring rounds) is 0,54 nm; diameter is 0,5 nm. Planes of neighboring peptide groups have the angle 105°, lateral radicals of a-amino acids are located on spiral external side, in other words, they are directed away from imaginary cylinder surface.

Main role at a-helix stabilization is played by hydrogen bonds which are formed between carbonyl oxygen atom of each first amino acid residue and hydrogen atom from –NH–group of every fifth amino acid residue (hydrogen bonds are formed between peptide bonds which are located at neighboring spiral rounds). Hydrogen bonds are practically parallel to a-helix axis. Usually protein chains are not completely spiral and a-helices alternate with linear parts.

Other secondary structure of poly-peptides is b-structure (pleated sheet or pleated layer). Elongated poly-peptide chains are located in pleated sheets. Chains are connected to each other by numerous hydrogen bonds between peptide groups.

Figure 2. Protein secondary structure: A – helix, B- pleated structure.

P rotein tertiary structure is determined by general poly-peptide chain space localization, which includes secondary structures elements. Globular (spherical) and fibrous (elongated threads) protein structures are differentiated.

Figure 3. Protein tertiary structure: globular and fibrous.

At tertiary structure formation poly-peptide chains are curved and lateral radicals of a-amino acid residues could react with each other, which are located too far from each other in un-curved chains. Several bond types play important role at protein tertiary structure formation and stabilization.

Hydrogen bonds appear between functional groups at lateral radicals (for example, between serine and threonine hydroxyl groups or between hydroxyl and carbonyl groups at glutamine and asparagine), and also between functional groups at lateral radicals and peptide bond atoms.

Ion bonds are formed due to electro-static interaction between ionic (able to form ion) radicals. For example, at aspartic acid radical there is carboxylic group which does not take part at peptide bond formation. At its dissociation negatively charged –СОО^– group is formed. At lysine radical there is amino group which does not take part at peptide bond formation. It could accept proton and acquire positive charge (–NH₃⁺). Between these charged groups ion bond is formed. Negatively charged group could be formed by glutamic acid, positively charged groups could be formed by arginine and histidine.

Hydrophobic interaction (non-polar bonds) appears due to mutual attraction of non-polar radicals (which do not contain functional groups) due to Van der Waals forces. This type of bond is typical of alanine, valine, leucine, isoleucine radicals, etc.

Disulfide bonds are formed between mercapto-groups which are at cysteine radical. –SH groups are easily oxidized with disulfide bridges formation. Disulfide bonds are especially numerous at keratine (hair and wool protein). Main bond types are presented at figure 4.

Figure 4. Formation scheme of ion (1) bonds, hydrogen bonds (2), hydrophobic bonds (3), disulfide bonds (4) between amino acid radicals at tertiary structure formation.

Protein quaternary structure is complex containing several poly-peptide chains. Each chain (sub-unit) keeps typical primary, secondary and tertiary structures. Quaternary structure is stabilized due to hydrogen bonds and hydrophobic interactions between sub-units. Protein with quaternary structure performs biological functions which are not typical of separate sub-units. Not all proteins are characterized by quaternary structure. An example of this protein is hemoglobin which main biological function is oxygen transition from lungs to organs and tissues. Quaternary structure of this protein is represented by four conjugated protein complex which comprises poly-peptide chain (globin) and non-protein part (hem).

Figure 5. Hemoglobin quaternary structure.

Question 57. What is protein classification?

Answer. There is no unified classification which takes into consideration different protein parameters. Usually classifications are based on one principle. Proteins could be classified according to:

– molecular form (globular and fibrous);

– molecular mass (low- and high-molecular);

– content or chemical structure (if there is non-protein part or not);

– functions;

– cellular localization (nucleus, cytoplasmic, lysosomal etc.);

– localization in the organism (blood proteins, hepatic proteins etc.);

– possibility to regulate these proteins amount: proteins with constant synthesis rate (constitutive) and proteins which synthesis rate could be increased due to external environment factors (inducible);

– life period in the cell (quickly renovated proteins with half-transformation period less than 1 hour, slowly renovated proteins with half-transformation period up to weeks and months);

– similar parts at primary structure and similar functions (protein families).

Globular proteins have relation between longitudinal and transverse axis which does not exceed 1:10, and in more often cases it is 1:3 or 1:4, i.e. protein molecule has the form of ellipse. Majority of human proteins belongs to globular proteins. They have compact structure, at globule formation hydrophobic radicals are directed inside the molecule, and hydrophilic radicals are on the globule surface, so their majority is well soluble in water. Examples are egg white (albumin), myoglobin, hemoglobin, many enzymes.

Fibrous proteins have elongated, thread-like structure, in which relation between longitudinal and transverse axis is more than 1:10. Fibrous proteins include collagen, elastin, keratine performing structural function at human body, and also myosin taking part at muscle constriction, and fibrin which is protein at blood coagulating system.

According to their structure all proteins are subdivided into unconjugated (simple) and conjugated (complex). Unconjugated proteins contain only a-amino acid residues. Conjugated proteins in addition to a-amino acid residues contain additional component with non-protein nature (prosthetic group). It could be organic substance, phosphoric acid, metal ion etc.

Proteins containing phosphoric acid residue in their structure (usually in the form of ester with serine hydroxyl group) are called phosphor-proteins: it is milk casein; vitellin, vitellinin and phosvitin, discharged from egg yolk; ovalbumin, discovered at egg white; ichtulin which is in fish caviar, etc.

Carbohydrates are prosthetic group at glycoproteins (these are components of blood serum - immunoglobulins, transferrins etc.); blood group substances; many viral antigens; some hormones and enzymes.

Lipids are in the structure of lipoproteins. Lipoproteins are subdivided into free or water soluble (blood plasma lipoproteins, milk, yolk lipoproteins etc.), and non-soluble or structural (cellular membrane lipoproteins, myelin cover of nervous tissues, chloroplasts at plants).

Nucleoproteins have nucleic acids: deoxyribonucleoproteins (DNP) and ribonucleoproteins (RNP).

Proteins perform many biological functions at cells. They could be subdivided into large groups:

– enzymes (specialized proteins catalyzing chemical reactions);

– regulating proteins (protein-hormones taking part at keeping internal organism medium constant, they act on special target-cells);

– receptor proteins (serve to transmit hormonal signal);

– transport proteins (take part at specific ligand transport from one organ to another, usually at complex with proteins molecules which are not dissolving in water are transported: blood plasma albumin transports fatty acid and bilirubin (hem degradation product) and erythrocyte hemoglobin transports О₂ from lungs to tissues etc.);

– structural proteins (give form, create support, define mechanic properties of a tissue: for example, tendon and cartilage main component is fibrous protein called collagen, which has high solidity; other structural protein (elastin) due to its structure provides particular tissues (vessels, lungs) property to stretch in all directions);

– protective proteins (for example, immunoglobulins have ability to recognize and bind foreign molecules, viral particles and bacteria as a result there is their neutralization; protective properties belong to proteins from blood coagulating system, for example, fibrinogen, thrombin: they take part at thrombus formation, which obstructs damaged vessel and prevents blood loss);

– constriction proteins (they give a cell ability to constrict or to move: actin and myosin are fibrous proteins taking part at skeletal muscle constriction; tubulin is important element, building material of cell organelles (micro-tubes which are important elements of cilia, with which help cells move).

There is a large number of proteins which have unique functions not covered at this classification.

Example is protein family: myoglobin family which includes, in addition to myoglobin itself, all types of hemoglobin.

Question 58. What qualitative reactions are used to identify amino acids and proteins?

Answer. General for all α-amino acids and proteins reaction is ninhydrin reaction which is blue-violet colour formation at amino acid or protein solution interaction with ninhydrin (organic reagent).

To find peptide bonds at peptides and proteins biuret reaction is used. It is red-violet colour formation at peptide or protein solution interaction with copper sulfate solution at alkaline medium. Violet colour could have different variations (red, blue). Reaction is determined by peptide bonds –СО–NН–, which connect amino acid residues. All peptides and proteins which have at least two such bonds, have positive reaction.

There are also specific reactions to find separate α-amino acids or relative amino acid groups. To find aromatic and hetero-cyclic α-amino acids (phenylalanine, tyrosine, histidine, tryptophan) xanto-protein reaction is used. For example, at action with nitric acid on amino acid tyrosine (or protein solution, in which structure there is tyrosine residue) nitro-compound is formed which is yellow. At adding alkaline solution colour is orange. At this reaction tyrosine aromatic radical is transformed, peptide bond is not affected.

Amino acids containing sulfur are found with the help of Fall’s reaction (with lead acetate). First protein solution is heated with concentrated alkaline solution (protein is partially hydrolyzed, sulfur is discharged in the form of ion S^2–), after that lead acetate solution is added (СН₃СОО)₂Pb and observe black precipitate formation of lead sulfide PbS:

(СH₃COO)₂Pb + 2 NaOH ® Pb(OH)₂ + 3 CH₃COONa

Pb(OH)₂ + 2 NaOH ® Na₂PbO₂ + 2 H₂O

Na₂PbO₂ + Na₂S + H₂O ® PbS¯ + 4 NaOH

If there is small amount of sulfur in the protein then instead of precipitate yellow-brown colour appears.

Sulfur ion S^2– could be found if to heated solution of protein and alkali we add sodium nitro-prusside solution Na₂[Fe(CN)₅NO]; there is red colour determined by complex salt formation:

Na₂S + Na₂[Fe(CN)₅NO] ® Na₄[Fe(CN)₅NOS]

Question 59. Is it possible to differentiate phenylalanine from tyrosine with the help of xanto-proteic reaction (with nitric acid)? If it is possible then what is the difference? If it is impossible explain why.

Answer. Xanto-proteic reaction is coloured reaction with its help it is possible to find amino acids with aromatic hydrocarbon radical, that is why, it is impossible to differentiate phenylalanine and tyrosine as in both of them there are benzene rings. At nitric acid action in both cases there is nitro-compound formed which has yellow colour.

Question 60. What amino acid was in the solution if at interaction with lead acetate in alkaline medium black precipitate was formed: a) alanine; b) serine; c) histidine; d) tryptophan; e) cysteine; f) tyrosine?

Answer. Reaction with lead acetate (Fall’s reaction) is aimed at sulfur containing amino acids. Among all enumerated α-amino acids only one amino acid cysteine contains sulfur. And it was in the solution.