Essay on Proteins. After reading this essay you will learn about 1. Classification of Proteins 2. Properties of Proteins and Amino Acids 3. Synthesis of Proteins and 4. Qualitative Tests for Plant Proteins and Amino Acids.
Essay on Proteins Contents:
- Essay on the Classification of Proteins
- Essay on the Properties of Proteins and Amino Acids
- Essay on the Synthesis of Proteins
- Essay on the Qualitative Tests for Plant Proteins and Amino Acids
Proteins and other related nitrogenous compounds are the principal constituents of protoplasm and hence are directly or indirectly involved in all the physiological processes taking place in the living cells. Evolution of life itself depended upon the pre-existence of the protein molecule.
As soon as the protein molecule was formed from inorganic materials, evolution of life became possible. Proteins often occur in plants in the form of stored foods, particularly in the seeds of many species. Such stored or reserve proteins differ markedly in their physical and chemical properties from the protoplasmic proteins which are highly complex and do not respond to the usual chemical protein tests.
The possible role of nucleic acids in protein synthesis has been analysed recently. All proteins contain carbon (50-54%), hydrogen (about 7%), oxygen (20-25%) and nitrogen (16—18%). All plant proteins contain sulphur; sulphurous amino acid, methionine, has an empirical formula, C5H11O2NS. Phosphorus is also a component of certain fundamental plant proteins.
The dimensions of protein molecules are enormous, the molecular weights of plant proteins usually ranging from a mere 10,000 to 6,000,000. Insulin, an animal protein (vertebrate hormone, secreted by pancreatic glands) is, however, the smallest known protein molecule, with a molecular weight of only 5,733 of 2 subunits each, i.e. = 11,466.
The molecular weights of virus proteins (nucleoproteins) of tobacco mosaic have been found to be in the neighbourhood of 40,000,000. Though the amino acid compositions of some proteins have been worked out, there are very few proteins again (exceptions insulin composed of 51 amino acids of 15 different types in two distinct chains and myoglobin) whose actual structure—spatial arrangement of amino acids—are known with certainty.
Proteins can be hydrolysed by treating them with acids or with suitable proteolytic enzymes. The end product of complete hydrolysis of any protein is always a mixture of amino acids.
During the course of hydrolysis a number of intermediate compounds of veiling complexities are formed:
Proteins → proteoses → peptones → polypeptides → dipeptides → amino acids.
It is evident, therefore, that the amino acids are structural units from which proteins are ultimately synthesised in the living cells.
Essay on the Classification of Proteins:
Because the molecular structure of most proteins are so imperfectly understood it is impossible to classify them on a strictly chemical basis. The physical properties such as solubility in acids, alkalis or salt solutions, coagulation, etc., are sometimes taken as basis of classification.
The following three main groups can be recognised:
I. Simple Proteins:
Yield only amino acids on hydrolysis. The more important simple proteins in plant cells are albumins, globulins, histories, prolamines, glutelins, etc. Albumins are characterised by their solubility in water; globulins are water-insoluble but readily dissolve in dilute salt solution. Prolamines and glutelins, which are soluble in 70% alcohol, are found in cereals.
II. Conjugated Proteins:
The members of this group are proteins with which are combined some non-protein groups. Examples are afforded by nucleoproteins (genes and viruses), phosphoproteins (casein of milk), chromoproteins (haemoglobin of red blood cells and phycobilins present in certain algae of the groups, Cyanophyta, Rhodophyta and Cryptomonad algae) and Lecithoproteins or lipoproteins (combination of protein with phospholipid, lecithin)—important constituents of protoplasm.
III. Derived Proteins:
This group includes products that are formed from partial hydrolysis or decomposition of simple proteins. Proteoses, peptones and peptides are representatives of the derived proteins.
Essay on the Properties of Proteins and Amino Acids:
Proteins are colloidal in nature and do not readily diffuse through plasma membranes. Almost all proteins are irreversibly coagulated by heat. Proteins are insoluble in neutral salts such as NaCl, MgSO4, etc., in which they precipitate without change in composition but go into solution on further dilution of the salt. Some are soluble in alkali while basic proteins are soluble in acids.
The amino acids are amphoteric compounds for the amino group is basic in reaction whereas the carboxyl group is acid; hence the amino acids are reactive and unite together to form larger molecules, the so-called peptide linkage—CONH—taking place between the amino and carboxyl groups with the elimination of water. Proteins consist of chains of amino acids ranging from a few too many, united by such peptide linkages.
Plant and animal protein molecules generally consist of about 200-1000 amino acid units. About 20 amino acids make up all proteins and all of them are characterised by the molecular grouping R—CHNH2COOH, with R standing for benzene or indole nucleus, an aliphatic chain or a sulphur-containing group.
All the amino acids found in plant proteins are of a-amino type.
The amino group, NH2 replaces hydrogen of one of the alkyl radical and the amino group is always attached to the so-called a-carbon atom which is next to the carboxyl group.
Unlike animals, plants must synthesise all the amino acids necessary for the formation of proteins. In addition, however, plants synthesise about 60 amino acids which, so far as is known, are not incorporated into protein.
Amides are salts of amino acids which correspond to inorganic salts. The two most commonly occurring amides in plants are asparagine and glutamine. Amides are formed from amino acids by replacement of hydroxyl part of the acid by another NH2 radical, e.g., asparagine. The amino acid concerned here is asparatic acid (derived from succinic acid). The amide glutamine is a derivative of the amino acid, glutamic acid which is obtained from the parent, α-oxoglutaric acid.
It is evident that amides contain more nitrogen compared to amino acids. In the green leaf, breakdown of proteins results directly or indirectly in the production of some amide, asparagine. Proteins are broken down to amino acids and the amino acids thus formed are converted to amides at the expense of respiratory energy (ATP molecules).
Essay on the Synthesis of Proteins:
Leaves must synthesise amino acids before they can make any proteins. If plants absorb inorganic nitrogen from soil in the form of NO–3 ions, the NO–3 ions must be converted into NH2 groups before being elaborated into amino acids. This would seem to indicate that NH+4 form of nitrogen would be more efficient source of nitrogen for the rapid formation of amino acids, since respiratory energy must be utilised to reduce NO–3 to NH+4 form.
Nitrate is reduced by a molybdoflavo-protein, nitrate reductase, the electrons for reduction being provided by NAD (P)H. The reduction of NO–3 to NH+4 form can also be accomplished by NADPH obtained from photolytic decomposition of water by chlorophyll. However, under certain conditions NO–3 form may be as satisfactory source of nitrogen as NO+4 form.
With the formation of NO+4 ion, the evidence indicates that it quickly combines with various keto acids—some of which are obtained from the Krebs cycle—to form amino acids. The synthesis of protein as also of fats is intimately linked with carbohydrate metabolism through the PSCR and Krebs cycles.
Specific enzymes are required for these synthesis. Amino acids are synthesised either in light or in darkness. In roots of some species of plants, synthesis of amino acids occurs in dark and they are translocated to the growing shoots where they are built up into proteins. In other plants, the inorganic nitrogen, either as NO–3 or NH+4 form is translocated directly to the leaves. The amino acids are synthesised in the leaves in presence of light and ultimately built up into proteins.
The synthesis of a particular protein requires that its constituent amino acids be arranged in a specific order. Recent theories postulate that the site of protein synthesis in the cell is capable of doing these because it continually receives information about the correct sequences of amino acids from the ultimate controller, the gene, situated in the chromosomes. The site of protein synthesis in prokaryotes is the 70S ribosome, in plants and other eukaryotes it is 80S. Each ribosome has two subunits, 30S and 50S in the case 70S particles, and 40S and 60S in the 80S particles.
The formation of peptide linkage involves the expenditure of energy; this is provided by ATP and GTP (guano- sine triphosphate). In the first step of protein synthesis amino acids are activated by ATP, to form aminoacyl adenylates—from which each amino acid is transferred to a specific type of RNA-molecules—small in size being about 78 nucleotides long, and soluble in water—each specific for a particular amino acid.
These RNA-molecules are transfer or soluble RNA (tRNA or sRNA). Unlike DNA these RNA molecules are single stranded, but are folded in the pattern of a clover leaf. The amino acids are hooked on at one end and the bulge at the opposite end is used for proper association with the messenger RNA at a particular region.
Biological information as is now known is coded in the four letter alphabet of nucleic acid bases—adenine, guanine, cytosine and thymine. The three dimensional chemical structure of adenine is such that it fits snugly only with thymine of DNA (or uracil of RNA), and guanine fits only with cytosine. Adenine is connected to thymine by a pair of hydrogen bonds; three H-bonds are formed between guanine and cytosine.
When RNA is synthesized by RNA polymerase, the sequence of nucleotides in the RNA, becomes exactly complementary to that in DNA, it thus is a negative replica of the nucleotide sequence in DNA. This RNA is considered to carry the message of DNA. This message has now to be translated into another language which is a 20-letter alphabet—in the form of 20 amino acids— permutations of which constitute different protein molecules.
This RNA now associates with a large number of 70S or 80S ribosomes—the polyribosomes or polysomes. For initiation and elongation and termination of the polypeptide chain several factors (which are themselves proteins) are required. The different factors in prokaryotes and eukaryotes are very similar. They are referred to as 1F1, 1F2, 1FS in so far as initiation of the protein chain is concerned. The mRNA associates with the 30S or 40S subunit first, with the help of 1F-2.
The first amino acid carried by a tRNA which initiates the polypeptide chain formation is methionine—usually formylated (formylated in prokaryotes and chloroplasts, not in plants); its tRNA has a sequence of UAC in the anticodon region and thus associates with the AUG region of mRNA. On each ribosome the tRNA attaches at the sites—one is called the P (peptidyl or donor) site, and the other A (or acceptor) site.
The amino acid carried by the tRNA on the P site is transferred to the next amino acid on the tRNA attached to the A site forming a peptide linkage between the free —NH2 and —COOH groups of the two amino acids. The tRNA on the donor site unloading its amino acid becomes free. The mRNA then moves like a conveyor belt bringing in a new amino acyl tRNA to the acceptor site, as the previous one occupies the P position. The peptide chain is then transferred to the newly arrived amino acid, and the process continues.
For elongation, elongation factors Ts and Tu are required in prokaryots, an EF-1 and EF-2 for plant systems. EF-1 stimulates the binding of amino acyl tRNA to ribosomes. The enzyme which forms the peptide bond is known as peptidyl transferase and translocation is achieved by EF-2, presumably a translocase. For translocation of peptidyl tRNA from A site to P site, another factor G is used.
The nucleotide sequence in the mRNA is specific for the nucleotide sequence in the anticodon region of the tRNA carrying a particular amino acid. It is now known that a three nucleotide sequence—a triplet—codes for a particular amino acid. Thus UUU codes for the amino acid phenylalanine, UGC for cysteine, AUG for methionine, and so on. The code is however, degenerate, though universal. Thus, leucine has 6 codons. i.e., there are 6 different tRNA molecules which can recognize leucine and carry it to the site of protein synthesis.
There are however, three nucleotide sequences UGA, UAA and UAG, which are not the codons for any amino acid, i.e., no tRNA can recognize it (unless mutated). Consequently when the elongating polypeptide chain comes to a point where one of these codons are there, the chain cannot elongate any further. However, the ribosome with the mRNA keeps moving resulting in the dislodgement of the polypeptide chain from the ribosome-aminoacyl-tRNA complex and it is set free. Some release factors R1, R2 and R3 are believed to be required for this. Then it becomes folded in a particular fashion to contribute to the three dimensional structure of the protein molecule.
The genetic code has been worked out largely through the effort or Nirenberg, Mathaei and Khorana. Nucleotide sequence of a tRNA was first achieved by Holley’s group. Marcus and associates have contributed considerably to our knowledge of protein synthesis in plants. The wheat germ system has been extensively investigated.
In some bacteria as in Bacillus, certain types of polypeptides are synthesized without the participation of ribosomes, mRNA and tRNA. Here the sequence is maintained by association of amino acids to a rnultienzyme complex in a specific manner followed by the formation of peptide linkage. The peptides may be cyclic and some amino acids are D-amino acids unlike L-amino acids used in ribosomal protein synthesis. Bacterial cyclic polypeptide antibiotics like bacitracin, gramiciden, polymyxin, etc. are synthesised in this way.
Step VII Chain propagation:
Repetition of steps V and VI for continued addition of amino acids to the carboxyl end of the growing polypeptide chain.
Schematic representation of the steps involved in ribosomal polypeptide synthesis (IF, initiation factors, (Peptidyl) n, peptide chain containing n aminoacids is given above.
An important feature of protein metabolism of the plant as a whole is the fact that the plant proteins appear to be in a state of continuous fluctuation wherein protein breakdown is balanced by resynthesize. Even if the total protein content of a leaf may remain constant, this certainly does not mean that the same individual protein molecules remain in the leaf.
On the contrary the decomposition of protein which is taking place continuously is more or less evenly balanced by protein synthesis. If we supply a mature plant, in which the total amount of protein in the leaves is relatively constant, with nitrogen in the form of NH+4 ions, it could be shown with the help of tracer technique, using 15N, that entirely new proteins have been synthesised. Since, however, total protein content remains constant, the new synthesis must have been balanced by an equal amount of protein breakdown.
A diagrammatic representation in broad outlines of protein synthesis and protein breakdown is given here:
Essay on the Qualitative Tests for Plant Proteins and Amino Acids:
Make extracts from crushed seeds, wheat flour, germinating seeds, etc. Take 10 g. of material, stir in 100 ml water or phosphate buffer and allow to stand for half an hour.
Filter and make the following tests with the filtrate:
I. Biuret test:
To a portion of the aqueous extract add about 1 ml of 4% NaOH soln. and a drop of 1 % CuS04 soln. A violet or pink colour is produced. This is a general test for all proteins containing the—GONH—group.
II. Xanthoproteic test:
To a portion of the aqueous extract add conc. HNOs (about 1/3 the vol.). A white ppt. is formed which on boiling may turn yellow and may also partially dissolve to give a yellow soln. Cool under the tap and add strong NH4OH till the reaction is alkaline. The yellow colour becomes orange. This test is given only by those proteins which contain the amino acids like tyrosine and tryptophan.
III. Millon’s test:
To about 5 ml of the protein extract, add about 1/2 its vol. of Millon’s reagent (5 grams of Hg in 95 ml conc. HNO3. Stir occasionally until brown fumes are no longer evolved. Add 200 ml water). A white ppt. is formed. On warming, the ppt. turns brick red, or it may dissolve, giving a red soln. Given only by the proteins having a phenolic grouping in the molecule, e.g., tyrosine.
IV. Sulphur test:
Boil about 5 ml of the plant extract with an equal vol. of 40% NaOH and a drop or two of 10% lead acetate. The soln. turns black or brownish due to the formation of PbS by the action of strong alkali on the sulphur of the protein [Na2S + (CH3COO)2 Pb = PbS + 2CH3COONa]. This is a positive test for the sulphur containing amino acids, e.g., cystine, methionine, etc.
V. Adamkiewicz’s reaction:
If conc. H2SO4 is added to a soln. of protein to which some acetic, or better, glyoxylic acid has previously been added, a violet colour is produced. The colour will appear as a ring at the juncture of the two liquids if H2SO4 is poured carefully down the side of the tube or throughout the mixture if shaken up. It depends upon the interaction of glyoxylic acid, which is generally present as an impurity in acetic acid, with the amino acid tryptophan and thus this test is given by all proteins containing tryptophan.
VI. Precipitation of proteins:
To a few ml of purified extract, add an excess of absolute alcohol. Protein is precipitated. Take about 5 ml aliquots of the extract in 3 test tubes and add respectively 5% CuSO4, 5% Pb-acetate and 5% HgCl2 soln.; protein is pptd. in each case. Proteins are also precipitated by (NH4)2 SO4, phosphotungstic acid, protamine sulphate, acetone etc.
Proteins are best separated by gel filtration using molecular sieves like Sephadex columns or chromatography on DEAE—cellulose, CM—cellulose or by gel electrophoresis.
B. Identification of Amino Acids:
By paper chromatography:
Mustard or legume seed, ground and covered with water, is incubated for about three days under toluene (to prevent bacterial infection) or extracted directly with 70% ethanol. 25 ml of this solution is pipetted off and filtered. Acidify the filtrate with acetic acid. Boil and filter off any further ppt. on cooling. Test aliquots of clarified extract for amino acids, tyrosine, tryptophan, arginine and cystine.
Evaporate a part of the clarified extract nearly to dryness and apply the solution at the corner of a filter paper. Develop the chromatogram with phenol/water and after drying, spray with 0.1% ninhydrin solution (the colour change due to reaction of ninhydrin with amino acids is utilised for the detection of the amino acids). Estimate the Rf value (the ratio of the distance traversed by the solute to that of the solvent front) of the spots and compare with spots containing known amino acids.
Specific tests for plant amino acids:
(a) Test for tyrosine:
Add a few drops of Millon’s reagent to the extract in a test tube. Take care to avoid excess. Heat and observe the gradual development of a dark red colour.
(b) Test for tryptophan:
(i) Add a few drops of glyoxylic acid and H2S04 to the extract in a test tube. Development of reddish-violet ring at the juncture of the two liquids is seen (see tests for proteins),
(ii) Add bromine water to another part of the extract. There is pink colouration.
(c) Test for arginine:
To 5 ml of the extract, add a few ml of a saturated solution of flavianic acid in water. A thick yellow ppt. is observed.
(d) Test for cystine:
(i) To a part of the extract, add sodium nitroprusside soln. and then a few drops of 40% NaOH. A conspicuous violet colour is developed,
(ii) Boil about 5 ml of the extract with a little of 40% NaOH and a few drops of 10% lead acetate solution. There is a black ppt. of lead sulphide (see test for proteins).