Practical Biochemistry E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Practical Biochemistry provides both foundational knowledge and advanced insights into biochemistry, including the basic compounds, and laboratory methods. The book is designed for students and academic professionals seeking a comprehensive understanding of the practical aspects of the subject.
The book is systematically divided into five sections, each dedicated to a specific category of macromolecules and related biochemical techniques: 1) Carbohydrates, 2) Proteins, 3) Nucleic acids, 4) Lipids, 5) Supplementary Techniques and Safety Data Sheet (SDS). Each chapter within these sections is structured to provide a thorough understanding of the aim, principles, procedures, and practical applications of biochemical techniques.
Key features:
· Comprehensive Information: meticulously organized and structured chapters provide a thorough and methodical approach to learning
· Additional Learning Tools: ‘Did You Know’ segments and ‘Viva Voice’ questions enrich the learning experience by offering interesting facts and stimulating critical thinking
· Practical Focus: Step-by-step guides aid readers in understanding and applying the techniques in the lab
· Safety and Accuracy: teaches how to conduct safe and accurate experiments with precautions
· Accessible Language: simple and lucid language helps beginners to understand complex biochemical concepts
Readership
This is a primary resource for biochemistry and other life science research courses at college and university levels that require learning practical laboratory techniques.
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Seitenzahl: 350
Veröffentlichungsjahr: 2024
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FOREWORD
Biochemical methods have contributed to diverse fields of science and technology over a century. They continue to be even more relevant today, half a century after molecular biology, genomics, and bioinformatics seem to hog the limelight. After all the genes are cloned and all the genomes are sequenced and analyzed bioinformatically, there is still no substitute for wet biochemistry to make progress in any area of modern biology or biotechnology. Apart from helping us in understanding the biological processes in any living organism, biochemical methods are critical in many applied branches such as medicine, agriculture, industrial microbiology, and biotechnology. Therefore, rigorous training in the protocols and practical skills of biochemistry greatly enhances the academic success and employability of students in every area of life sciences and biotechnology. This necessitates constant upgradation of books and manuals, and this book is the latest effort in that direction.
"Practical Biochemistry" edited by Dr. Pamela Jha is a comprehensive guide designed for students and early researchers in the whole gamut of life sciences. It encompasses various disciplines such as cell biology, molecular biology, and medical sciences, offering a holistic understanding of biochemistry through its underlying methods. The book rightly emphasizes both theoretical foundations and practical protocols, catering to individuals at the bachelor's and master's levels, as well as research scholars. Each experiment is accompanied by a quick recap of the relevant theory, including specific reactions and protocols, supplemented by 'viva voce questions' for interactive learning. Expected results with quantitative values are provided for many experiments, aiding in self-verification and comprehension. Dr. Pamela Jha's book is structured for quick and effective revision, making it a valuable resource for students and enthusiasts alike. Its user-friendly approach and comprehensive coverage ensure an enjoyable learning experience in the field of biochemistry.
I congratulate Dr. Jha and her team of authors and the publisher for this book.
PREFACE
The first edition provides information that is important for understanding of some basic and advanced biochemical methods for qualitative and quantitative estimation of major macromolecules. This book is organized into 5 sections, i.e., carbohydrates, proteins, nucleic acids, lipids, and other techniques and SDS. Each section comprises multiple chapters structured with abstract, keywords, introduction, aim, principle, requirements, procedure, probable dummy results, conclusion, precautions, and finally, references. In addition, each chapter is also supplemented with relevant interesting facts in the ‘did you know’ segment and ‘viva voice’ questions. I have attempted to write this book in a simple and lucid language to enable easy, focused learning.
I welcome constructive comments from students and readers. Finally, I express my gratitude to the contributors and publisher who have extended tremendous support in the production of this first edition. I wish everyone happy learning!
List of Contributors
REFERENCES
[1]Nelson, D.L.; Lehninger, A.L.; Cox, M.M. Lehninger principles of biochemistry.; Macmillan, 2008.[2]Berg, J.M.; Tymoczko, J.L.; Stryer, L. Biochemistry (Loose-Leaf).; Macmillan, 2007.[3]Jain, J.L. Fundamentals of biochemistry. S.; Chand Publishing, 2004.[4]Bartnik, M; Facey, PC Glycosides. In Pharmacognosy; Academic Press, 2017. pp. 101-161.Qualitative Tests for Carbohydrate Detection
Mrittika Sarkar1,Sinchan Hait2,*,Sai Joshi3Abstract
Qualitative tests for the determination of carbohydrates in the given sample are a crucial part of before getting into major analytical procedures. Every mono-, oligo-, and poly-saccharide vary depending upon the number of carbohydrate molecule present in it as well as changes in the side chains. Thus, every carbohydrate molecule has distinct functions and properties. Depending upon these physio-chemical properties of the carbohydrates, they respond to certain specific chemical reactions under certain conditions. Only mono- and dis-saccharides respond to the solubility test as they are soluble in water at room temperature. The Molisch test is only for the determination of the presence of carbohydrate, not depending on the types of it. The iodine test gives a result for polysaccharides. Whereas Fehling, Benedict and Osazone tests distinguish between reducing and non-reducing sugars. The Bradford test differentiates between mono- and di-saccharide-reducing sugars. Seliwanoff test is only for sucrose which is a non-reducing sugar. Bial’s test is to determine the presence of pentose sugars.
INTRODUCTION
To identify the presence of carbohydrates, if present or not in an unknown sample, a certain standardised preliminary protocol is to be followed. There have been various ways to qualitatively determine of carbohydrate present in a given sample. These tests are based upon the physico-chemical properties of the carbohydrates: mono-, oligo-, and polysaccharide; and reducing and non-reducing sugars can be differentiated. Detection and characterizationof carbohydrates present in an unknown sample solution based on various qualitative chemical assays are discussed below (Fig. 2.1).
Solubility Test
Aim
To determine the type of carbohydrates present in each sample by solubility test.
Fig. (2.1)) Summary of all qualitative tests for carbohydrates.Principle
Both monosaccharides (e.g., glucose) and disaccharides (e.g., sucrose) are crystalline solids at room temperature, but they are soluble in water because each molecule has several -OH groups that readily engage in hydrogen bonding with water molecules. Whereas in the case of polysaccharides, they have a complex structure which has strong interaction among polysaccharide molecules via hydrogen bonds, so they are insoluble in water [1, 3].
Requirements
Glasswares
• Test tubes
Other Requirements
• Dropper
• Test tube holder
Reagents
• Sugar solution (5% glucose, 5% sucrose, 1% starch)
• Distilled water
Procedure
To 1ml of sugar solution, add a few drops of distilled water and mix well.
Applications
• This test can help to determine whether mono-disaccharides or polysaccharides are present in the solution and their ability to dissolve in water.
Conclusion
If it solubilizes, then it confirms the presence of monosaccharides or disaccharides (glucose, sucrose) or else the presence of polysaccharides (starch).
Molisch’s Test
Aim
To determine whether carbohydrates are present in a given sample by the Molisch test.
Principle
The Molisch test is a qualitative test to detect carbohydrates. Mainly monosaccharides, disaccharides, polysaccharides and glycoproteins (biomolecule complex of protein and sugar) can be determined using this test. The test has been named after Czech- Austrian botanist Hans Molisch.
When monosaccharide is treated with conc. H2SO4 or conc. HCl, OH groups of sugar are removed in the form of water, after which furfural is formed from pentose sugar and hydroxymethyl furfural from hexose sugar (Fig. 2.2). These products (furfural and hydroxymethylfurfural) react with sulphonated α- naphthol to give a purple or violet-coloured complex at the interface of the acid and test layer (Fig. 2.2) [1, 2]. If the test sample contains a poly- or disaccharide, a glycoprotein or glycolipid, the acid will first hydrolyse them into monosaccharide components, after which they get dehydrated to form furfural or its derivatives.
Fig. (2.2)) Formation of purple or violet-coloured complex.Requirements
Chemicals
• Test solution (5% glucose solution)
• Molisch’s reagent (Add 5% (w/v) α-naphthol in 95% ethanol or dissolve 500 mg α-naphthol in 10 ml 95% ethanol)
• Conc. H2SO4
Glasswares
• Test tubes
• Pipette (1 ml Beral pipette)
Other Requirements
• Test tube holder
• Dropper
• Distilled water
Procedure
1. Take 2ml of test solution in a dry test tube.
2. As a control take 2ml of distilled water in another tube.
3. Add 1-2 drops of Molisch’s reagent to both tubes.
4. Gently pipette 1ml conc. H2SO4 and pour it along the side of the tube to get two distinct layers.
5. Observe the change in colour at the junction of two layers.
Conclusion
A purple colour between the junction of 2 layers indicates the presence of carbohydrates in the sample. No appearance of the purple complex indicates a negative test. However, if the appearance of green ring observation occurs, then it indicates that other impurities are present in the reagent, which interacts with α-naphthol and the conc. acid. The presence of concentrated sugar solution in the test sample yields a red ring due to partial charring of the sugar by the acid.
Precautions
• Do not pour conc. sulfuric acid directly into the solution as it can cause charring of carbohydrates due to heat generated during the reaction, which will result in a black ring, giving a false negative test.
• The α-naphthol solution should be prepared fresh as it is unstable.
Applications
This test can be used most practically to detect the presence of sugar in food products that claim to be sugar-free.
Fehling Test
Aim
To determine whether reducing sugars are present in a given sample by Fehling’s test.
Principle
This test is used to differentiate between water-soluble carbohydrate and ketone functional groups and can be used supplementary to the Tollens' reagent test. It was developed by German chemist Hermann von Fehling.
Fehling’s test is one of the sensitive tests for the detection of reducing sugars. Fehling’s test is given positive by reducing sugars containing specifically aldehyde and alpha-hydroxy ketone groups. Fehling’s reagents consist of two solutions: Fehling's solution A, which is aqueous copper sulphate and Fehling’s solution B, sodium potassium tartrate (Rochelle salt), a strong alkali. This Fehling’s B solution present in the reagent acts as the chelating agent in this reaction. Both Fehling A and B solutions are mixed in equal amounts to make the Fehling reagent.
On heating the sample with Fehling’s reagent, aldehydes oxidize, giving a positive result, but ketones do not unless they are α-hydroxy ketones. The bis-tartrato cuprate(II) complex oxidizes the aldehyde to a carboxylate anion or aldonic acids, and in the process, the copper(II) ions of the complex are reduced to copper(I) ions. Red copper(I) oxide then precipitates out of the reaction mixture, which indicates a positive result (Fig. 2.3) [1, 2]. The ketoses are oxidized to yield shorter chain acids.
Fig. (2.3)) Formation of cuprous oxide (red precipitate).Requirements
Chemicals
• Test solution (5% glucose or 5% sucrose)
• Fehling reagent (Fehling's “A”: Dissolve 7 g CuSO4. 5H2O in distilled water containing 2 drops of dilute sulfuric acid. Fehling's “B”: Add 35g of potassium tartrate and 12g of NaOH in 100 ml of distilled water. Mix both A and B in equal quantities to make Fehling’s reagent).
Glasswares
• Test tubes
• Pipette (1 ml Beral pipette)
Other Requirements
• Test tube holder
• Water Bath (100 °C)
Procedure
1. Take a test tube and add 5 ml of the test solution to it.
2. Add Fehling’s reagent to this test tube.
3. Shake the test tubes properly and gently place them into boiling water for a water bath.
4. Carefully observe the test tubes and note down the result of each test tube after 5 minutes.
Conclusion
The appearance of a reddish-brown precipitate indicates a positive result, thus the presence of reducing sugar (e.g., Glucose, fructose, lactose). And the appearance of blue colour and no precipitate is the presence of non-reducing sugars (e.g., sucrose, starch).
Precautions
• Fehling's solution is corrosive and toxic, wear appropriate personal protective gear while performing this experiment.
Applications
• It is used to screen for glucose in the urine, thus detecting diabetes.
• This test is used in the breakdown of starch to glucose syrup and maltodextrins, to measure the amount of reducing sugar, thus revealing the dextrose equivalent (DE) of the starch sugars.
Benedict’s Test
Aim
To determine whether reducing sugars are present in a given sample by Benedict’s test.
Principle
There are generally two types of sugar: reducing (all monosaccharides, as they have free anomeric carbon in their structure, i.e., free aldehyde or ketone group that can reduce cupric salt) and non-reducing (some disaccharides and polysaccharides). Benedict’s reagent is sensitive and can be used to determine and demonstrate the reducing property of carbohydrates (even to small quantities of reducing sugars (0.1%)) and was discovered by Stanley Rossiter Benedict, an American chemist [2].
When reducing sugar is heated with Benedict’s reagent, due to the presence of the alkaline sodium carbonate, the reducing sugar gets converted into a strong reducing agent called enediol, which decreases the cupric particles (Cu2+) present in the reagent to cuprous particles (Cu+), which appear as insoluble red copper oxide (Cu2O) (Fig. 2.4) [1, 2]. This is a semi-quantitative test, as the colour of the precipitate can indicate the approximate quantity of sugar present in the sample.
Fig. (2.4)) Formation of cuprous oxide (brick red precipitate).Requirements
Chemicals
• Sample solution (5% glucose or 5% sucrose)
• Benedict’s reagent (A deep-blue alkaline chemical reagent which consists of copper sulphate pentahydrate (CuSO4.5H2O) in the Citrate buffer. Dissolve 17.3 g sodium citrate and 10 g sodium carbonate in 85 ml H2O. Filter it. Dissolve 1.73 g CuSO4.5H2O in 10 ml H2O, followed by the addition of carbonate-citrate mixture).
Glasswares
• Test tubes
• Pipette
Other Requirements
• Test tube stand and holder
• Hot water Bath
• Bunsen burner
Procedure
1. Pipette out 2 ml (10 drops) of Benedict’s reagent and place it in the clean test tube.
2. Add approximately 1 ml of sample to Benedict’s reagent.
3. Water bath this sample for 3-5 minutes (can be heated directly over a flame).
4. Observe for precipitate formation in the test tube.
Conclusion
A reddish precipitate within three minutes indicates the presence of reducing sugars present. Example: Glucose. No precipitation indicates the absence of reducing sugar and probably non-reducing sugar. Example: Sucrose.
Precautions
1. Benedict's solution is an irritant, so avoid contact with the skin and eyes.
Applications
Benedict's test can be used to detect the presence of glucose (reducing monosaccharide) in urine.
Seliwanoff’s Test
Aim
To detect the presence of aldose and ketose sugar by Seliwanoff’s test.
Principle
Seliwanoff’s test is used to differentiate between aldose and ketose sugars, and it is specific for ketohexoses. This test is very similar to Bial’s test.
When the sample is heated with concentrated acid, samples with ketoses are dehydrated more rapidly to give furfural derivatives, and condensation with resorcinol gives cherry red complex (Fig. 2.5) [1, 2]. If the reaction is allowed for a longer time (more than 10 minutes) for samples with aldoses, it may also produce positive results as the reaction with aldehydes takes place slowly. Seliwanoff’s test can distinguish between two monosaccharides, e.g. glucose (aldehyde group present) and fructose (ketone group present).
Fig. (2.5)) Formation of red cherry colour.Requirements
Chemicals
• Test solution (5% fructose or 2% glucose)
• Seliwanoff’s reagent (Dissolve 110 mg of resorcinol in 220 ml of 3N HCl or dissolve 50 mg resorcinol in 33 ml conc. HCl, then make it up to 100 ml with H2O).
Glassware
• Pipettes
• Test tubes
Other Requirements
• Water bath
• Test tube holder
Procedure
1. Add 5 ml of Seliwanoff’s reagent and 1 ml of a sample in a test tube.
2. Heat the solution using a water bath in boiling water for 1-5 minutes and observe for colour change.
Conclusion
A positive test is indicated by the colour of the sample turning to cherry red immediately, which means keto sugar is present in the solution (e.g., fructose). If the red cherry complex is not formed immediately, then wait for about 10 minutes; if a slower framing pink colour is seen, then aldehydes are present, indicating a positive test (e.g., glucose). Otherwise, it is a negative test (No colour is seen.)
Precautions
• Make sure to add 5 ml of reagent and 1 ml of material to be tested because this is for the exact compatibility of both agents.
• Make sure that what’s inside the test tube never contacts anyone because it is highly corrosive and can bring serious damage to the skin.
