Thursday, April 23, 2015

Salivary amylase and starch

Salivary amylase is a digestive enzyme secreted by the salivary glands, and about 1.7 liters of saliva is produced everyday by a healthy human. It is responsible for breakdown of the polysaccharide “starch” into dextrins (oligosaccharides), maltose (disaccharide) and glucose (monosaccharide) in the mouth.


Starch consists of two types of molecules, “amylose” (20-30%) and “amylopectin” (70-80%) and both are polymers of glucose. Amylose is a linear chain of glucose units that are linked by α-1,4 bonds, whereas amylopectin is a branched molecule in which glucose units are linked by α-1,4 bonds in the straight chain region in addition to α-1,6 bonds at the branching points. The relative proportions of amylose to amylopectin depend on the source of the starch.

Branch of amylopectin

Salivary amylase hydrolyses only the α-1,4 bonds that link glucose units. Therefore, starch and glycogen, which consist of α-1,4 bonds, are hydrolyzed by amylase while. The cellulose consisting of β-1,4 bonds, cannot be hydrolyzed by salivary amylase. The α -amylase constitute a family of endo-amylases that catalyze the initial hydrolysis of starch into shorter oligosaccharides through the cleavage of α-D-(1-4) glycosidic bonds. Neither terminal glucose residues nor α-1,6-linkages can be cleaved by α-amylase.

The conformation of (α-1→4) linkages in amylose, amylopectin causes these polymers to assume coiled helical structures. The iodine ion will be trapped in the coiled helical structures of starch and gives blue color. After digestion of starch by salivary amylase, no more helical structures available, and no color will be produced in addition of iodine. Therefore iodine can be taken as indicator to measure the activity of amylase, and sometimes, to find the inhibitor of amylase.

Nelson DDL, Cox MM (2005) Lehninger Principles of Biochemistry, 5th edition, W.H. Freeman and Co. Publisher, New York, USA, 248-252.

Wednesday, April 15, 2015

Model Questions for Biochemistry-II

Model Questions for Biochemistry (Microbiology)

Long Answer Question: (5 × 5 = 25)
1) Explain the biological functions of proteins with examples.
2) Explain the essential amino acids.
3) What is the transamination process? Mention its significance.
4) What are the nucleotides? Explain the biological roles of nucleotides.
5) What are the denaturation and renaturation processes of DNA?

Multiple Choice Question: (1 × 5 = 5) (any five)
1. Marfan's syndrome is thought to be a mutation affecting
A. hemoglobin synthesis                       B. insufficient thyroid production
C. metabolism of homogentisic acid     D. collagen and/or elastin synthesis

2. Protein folding is
A. automatic, mediated by the protein itself
B. mediated by other proteins called chaperones
C. mediated by the ribosomes
D. by the protease enzyme

3. The isoelectric point of an amino acid is defined as the pH
A. where the molecule carries no electric charge
B. where the carboxyl group is uncharged
C. where the amino group is uncharged
D. of maximum electrolytic mobility

4. What is the heaviest of the twenty amino acids?
A. phenylalanine             B. tryptophan
C. tyrosine                       D. histidine

5. D-Alanine and L-Alanine are technically known as
A. polymer                       B. enantiomers
C. epimers                        D. anomers

6. Which of the following pairs of amino acids would carry a negative charge on their side chain at pH 8.0?
A. leucine & glycine              B. asparagine & glutamine
C. histidine & lysine              D. aspartate & glutamate

7. Which of the following amino acid do not fall under the category of essential amino acid?
A. leucine             B. histidine            C. glycine           D. methionine

8. What is the end product of leucine metabolism?
A. acetyl-CoA                            B. pyruvic acid
C. oxaloacetic acid                    D. acetyl carnitine

9. The nitrogen atoms of urea produced in the urea cycle are derived from
A. ammonia                                   B. ammonia and aspartic acid
C. nitrite                                         D. nitrate

10. The products of urea cycle are
A. 1 molecule of urea, 1 molecule of ammonia, 3 molecules of GTP and 1 molecule of fumaric acid
B. 1 molecule of fumaric acid, 1 molecule of urea, 1 molecule of AMP, 2 molecules of ADP
C. 1 molecule of aspartic acid, 1 molecule of ammonia, 1 molecule of fumaric acid, 1 molecule of ATP
D. 1 molecule of urea, 1 molecule of ammonia, 1 molecule of ATP and 1 molecule of fumaric acid

11. Which of the following is used as carbon atom source while producing urea in the urea cycle?
A. arginine                                         B. aspartic acid
C. carbon dioxide                               D. glucose

12. Urea cycle converts
A. ammonia into a less toxic form
B. urea into nitrogen
C. amino acids into ketoacids
D. ketoacids into amino acids

13. The inputs to one cycle of the urea cycle are
A. 1 molecule of aspartic acid, 1 molecule of ammonia, 1 molecule of carbon dioxide, 3 molecules of ATP
B. 1 molecule of urea, 1 molecule of ammonia, 3 molecules of ATP and 1 molecule of fumaric acid
C. 1 molecule of fumaric acid, 1 molecule of urea, 3 molecules of AMP
D. 1 molecule of acetic acid, 1 molecule of urea, 3 molecules of GTP

Model Questions for Biochemistry

Model Questions for Biochemistry (Biotechnology)

Long Answer Question: (6 × 1 = 6) (any one)
1) Explain the biological functions of proteins with examples.
2) Explain the essential amino acids.

Short Answer Question: (2.5 × 2 = 5) (any two)
1) Describe how peptide bond is formed.
2) Draw the structures of non-polar and non-charged aliphatic amino acids.
3) Mention the the enzymes and co-factors involved in hydroxylation of proline and lysine in collagen synthesis.

Very Short Answer Question: (1 × 4 = 4) (any four)
1) Define denaturation of proteins.
2) Which bonds are flexible for rotation in a dipeptide?
3) Mention the replaced amino acids that leads to sickle cell anemia.
4) Define the quaternary structure of proteins?
5) What are the major amino acids in collagen?
6) What is the Ramachandran Plot?

Multiple Choice Question: (1 × 5 = 5) (any five)

1. Marfan's syndrome is thought to be a mutation affecting
A. hemoglobin synthesis                       B. insufficient thyroid production
C. metabolism of homogentisic acid     D. collagen and/or elastin synthesis

2. Protein folding is
A. automatic, mediated by the protein itself
B. mediated by other proteins called chaperones
C. mediated by the ribosomes
D. by the protease enzyme

3. The isoelectric point of an amino acid is defined as the pH
A. where the molecule carries no electric charge
B. where the carboxyl group is uncharged
C. where the amino group is uncharged
D. of maximum electrolytic mobility

4. What is the heaviest of the twenty amino acids?
A. phenylalanine             B. tryptophan
C. tyrosine                       D. histidine

5. D-Alanine and L-Alanine are technically known as
A. polymer                       B. enantiomers
C. epimers                        D. anomers

6. Which of the following pairs of amino acids would carry a negative charge on their side chain at pH 8.0?
A. leucine & glycine              B. asparagine & glutamine
C. histidine & lysine              D. aspartate & glutamate

7. Which of the following amino acid do not fall under the category of essential amino acid?
A. leucine             B. histidine            C. glycine           D. methionine

Wednesday, April 01, 2015

Qualitative Detection of Glucose in Urine

Glucose is not present in urine, in general because in the kidneys, glucose is reabsorbed from the filtrate of glomerulus, across the tubular epithelium of proximal tubule into the bloodstream. Therefore, normal glucose range in urine is 0 mg/dL, or rarely up to 15 mg/dL. If higher than normal levels of glucose (0-15 mg/dL) is found in urine, then it can be suspected as diabetes, pregnancy, or renal glycosuria. Presence of glucose in urine was commonly tested to monitor diabetes in the past. Now, blood tests for glucose is easier than urine test. The glucose urine test may be done when there is suspect of renal glycosuria.

One of the test is based on the basis of character of glucose as it is a reducing carbohydrate because of a free aldehyde group. During the reduction process of Cu(OH)2 to CuOH by the glucose aldehyde group, and itself is converted into a carboxyl group. CuOH and its degradation product, Cu2O, are colored compounds. In excess of CuSO4, the high Cu(OH)2 content may lead to the formation of black CuO particles. This is called Trommer’s reaction and explained in the following equations;

Trommer's Explannation
Karl Trommer (1806-1879), German chemist first developed this copper reduction test in 1841, and more stable copper sulphate reagent was explained by Hermann von Fehling (1812-1885) in 1850. Even more sensitive copper reagent was described in 1908 by Stanley Benedict (1884-1936). Later, Ames (1945), the research team led by Albert & Helen Free, developed Clinitest tablet, which contained cupric sulfate, sodium hydroxide, and citric acid mixed with a bit of carbonate to make it fizz. Helen M. Free (1923-) and Alfred Free (1913–2000), revolutionized diagnostic urine testing with their invention of a chemically coated paper dipstick that measures a patient’s blood/urine sugar by changing color when dipped in a blood/urine sample.

Tuesday, March 24, 2015

Determination of protein concentration

Introduction
Finding the exact quantity of proteins in a solution is very often necessary in the biochemical practice and to analyse clinical samples as well as in research. There are many ways to measure protein concentration. In chromogenic methods, the absorbance of a coloured product formed by the protein and an organic molecule is measured. Protein concentration can also be determined from the protein's own (intrinsic) UV absorbance. However, these methods may give different results for different proteins of the same concentration. Also, different methods can yield somewhat different results for the same protein. Also, dilution factor may be a important parameter.

Text source
There is no absolute photometric protein concentration assay. All methods have advantages and disadvantages and we must choose among them by taking the following aspects into consideration: specificity, sensitivity, the measurable range of concentration, the accuracy, the nature of the protein to be examined, the presence of materials interfering with the measurement, and the time required for the measurement.

Principle

Biuret test
Molecules with two or more peptide bonds react with Cu2+ ions in alkaline solution and form a purple complex. Nitrogen atoms of the peptide bonds form a coordination bond with the metal ion. The quantity of the complexes formed is proportional to the number of peptide bonds.

In practice, the determination of protein concentration is done using a calibration curve created using samples of known concentration. The protein treated with biuret reagent is measured at 540 nm after the purple product is formed.

The advantages of the method include that only few materials (e.g. Tris and amino acid buffers) interfere with it, it can be done in a short time and does not depend on the amino acid composition of the protein. Its disadvantages are its low sensitivity and that it requires at least 1 mg of protein.

Lowry (Folin) protein assay
This is sensitive technique where a coloured product is formed similarly to the biuret reaction, but a reagent strengthen the colour, which is kown as Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction). The reaction mechanism is not well understood, but involves reduction of the Folin–Ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine). The strong blue colour is created by two reactions: (1) formation of the coordination bond between peptide bond nitrogens and a copper ion and (2) reduction of the Folin-Ciocalteu reagent by tyrosine (phosphomolybdic and phosphotungstic acid of the reagent react with phenol). The measurement is carried out at 750 nm.

As in the biuret reaction, a calibration curve is created (for example using BSA, bovine serum albumin), and the concentration of the unknown protein is determined from the curve.

The advantages of the method include that it is quite sensitive and is able to detect even 1 µg of protein. Its disadvantages are that it takes rather long to carry out, is disturbed by various materials (including ammonium sulphate, glycine and mercaptans) and that the incubation time is critical. As different proteins contain different amounts of tyrosine, the amount of the coloured product will also be different. As a consequence, this method is more suited to compare the concentration of solutions of the same protein than to absolute measurement.
Source:

Monday, March 23, 2015

List of Experiments for Biochemistry Laboratory work

                        List of Experiments for Laboratory work
Course Title: Laboratory work I: Biochemistry (Microbiology)
Credit: 1                                                                                                                                  

1. Preparation of normal, molar and percent solutions.
2. Preparation of different buffer solutions and measure their pH.
3. Determination of the isoelectric point of casein (of milk).
4. Titration of amino acids and find the physical constants.
5. Study the effects of temperature and pH on the enzymatic activity of salivary amylase.
6. Estimation of given protein concentration by Bradford, Lowry (Folin) protein assay and by Biuret test.
7. Qualitative detection of glucose in urine (the Trommer reaction).
8. Quantitative determination of glucose in blood by a glucose-oxidase method.
9. The determination of activity of the Krebs cycle dehydrogenases in the liver.
10. Determination of total cholesterol concentration in serum.
11. Determination of urea concentration in serum and urine.
12. Quantitative determination of phospholipids concentration in serum.
13. Determination of saponification value of the fat.
14. Separation of amino acids by chromatographic techniques and detection by nin-hydrin test. 

Tuesday, March 12, 2013

A Strategy Game in Linux Environment, 0 AD (zero-ey-Dee)

If you are fond of strategy game, 0 A.D. (zero-ey-dee) is one of the best game, which is available in Linux (also in Windows and Mac). It is a free, open-source, historical Real Time Strategy (RTS) game.

The game winning secrets resembles with the “age of empire 2,” i.e., build more and more modern weapons and attack as soon as possible. Defensive strategy hardly works.

However, currently it is available only in alpha version, by Wildfire Games. Even though it is in alpha phase. I have tested and it is working well in Ubuntu 12.04 LTS except the movement became very slow in some missions.

0 AD provides a platform not only for playing game, but also we can build a scenario and map ourselves. Here are some print-screens.







Monday, March 04, 2013

Is Debian the Most Stable Linux Distro?

I have been using Fedora for many years. Fedora is the leader out-breaking new software. I have found many research related software are more comfortable in fedora than some other Dostros, which I have tested. But the greatest drawback of this OS is the instability and crashing of several times. Surprisingly windows 7 showed great improvement, however, it is not freeware.

Because of YUM and RPM system and is bluish configured, I had shifted in CentOS. Although CentOS is designed for server, it was good in desktop also. And I could install research related software and they were working well, however, not good for Skype. But when I made update a few days after installing 6.3, the system did not start. There was no facility for bug report at that case. It booted only and hanged on log in screen. I had restarted several times but same problem persisted.

Then I tried Linux Mint. I surprised with its awesome feature. Really, it was very good looking. But yesterday when I updated Linux Mint 14, Nadia, I got the same problem as CentOS. Does it mean Linux OS are still in infancy in terms of reliability and stability.

Probably, I have to try Debian but may I install the latest version of some software such as Libreoffice, VLC, Autodock, Chimera, etc.

At this stage I need such Distro, which is stable for at least for 2-3 years without compromising for the latest software and security. I mean the software / application could be installable a few months later than the final release date, if not immediately. Please comment if you have any idea.