Monday, February 22, 2010

Harper's Biochemistry 28th edition

Harper's Illustrated Biochemistry, 28th Edition : McGraw-Hill Professional Online Book Store

Overview

The biochemistry text that every medical student must own--now in full color!

Comprehensive, concise, and up-to-date, Harper's is unrivaled in its ability to clarify the link between biochemistry and the molecular basis of health and disease.

The Twenty-Eighth Edition has undergone sweeping changes -- including a conversion to full-color artwork and the substantial revision and updating of every chapter -- all to reflect the latest advances in knowledge and technology and to make the text as up-to-date and clinically relevant as possible. Combining outstanding full-color illustrations with integrated coverage of biochemical diseases and clinical information, Harper's Illustrated Biochemistry offers an organization and clarity not found in any other text on the subject.

Striking just the right balance between detail and brevity, Harpers Illustrated Biochemistry is essential for USMLE review and is the single best reference for learning the clinical relevance of a biochemistry topic.

NEW to this edition:

* Full-color presentation, including 600+ illustrations
* Every chapter opens with a Summary of the Biomedical Importance and concludes with a Summary reviewing the topics covered
* Two all-new chapters: "Free Radicals and Antioxidant Nutrients" and "Biochemical Case Histories" which offers an extensive presentation of 16 clinical conditions
* A new appendix containing basic clinical laboratory results and an updated one with a list of important websites and online journals
* NEW or updated coverage of important topics including the Human Genome Project and computer-aided drug delivery

Table of contents

1. Biochemistry and Medicine
2. Water & PH
3. Amino Acids and Peptides
4. Proteins: Determination of Primary Structure
5. Proteins: Higher Orders of Structure
6. Proteins: Myoglobin and Hemoglobin
7. Enzymes: Mechanisms of Action
8. Enzymes: Kinetics
9. Enzymes: Regulation of Activities
10. Bioenergetics: The Role of ATP
11. Biologic Oxidation
12. The Respiratory Chain & Oxidative Phosphorylation
13. Carbohydrates of Physiologic Significance
14. lipids of Physiologic Significance
15. Overview of Metabolism
16. The Citric acid Cycle: The Catabolism of Acetyl-CoA
17. Glycolysis and Oxidation of Pyruvate
18. Metabolism of Glycogen
19. Gluconeogenesis and Control of the Blood Glucose
20. The Pentose Phosphate Pathway & other Pathways of Hexose Metabolism
21. Biosynthesis of Fatty Acids
22. Oxidation of Fatty Acids: Ketogenesis
23. Metabolism of Unsaturated Fatty Acids & Eicosanoids
24. Metabolism of Acyglycerols and Sphingolipids
25. Lipid Transport And Storage
26. Cholesterol Synthesis, Transport and Excretion
27. Integration of Metabolism- the Provision of Metabolic Fuels
28. Biosynthesis of Nutritionally Nonessential Amino Acids
29. Catabolism of Proteins & of Amino Acid Nitrogen
30. Catabolism of the Carbon Skeletons of Amino Acids
31. Conversion of Amino Acids
32. Porphyrins & Bile Pigments
33. Nucleotides
34. Metabolism of Purine & Pyrimidine Nucleotides
35. Nucleic Acid Structure and Function
36. DNA Organization, Replication and Repair
37. RNA Synthesis, Processing and Modifications
38. Protein Synthesis and Genetic Code
39. Regulation of Gene Expression
40. Molecular Genetics, Recombinant DNE and Genomic Technology
41. Membranes: Structure and Function
42. The Diversity of Endocrine System
43. Hormone Action and Signal Transduction
44. Nutrition, Digestion and Absorption
45. Vitamins and Minerals
46. Intracellular Traffic and Sorting of Proteins
47. Glycoprotein
48. The Extracellular Matrix
49. Muscle and the Cytoskeleton
50. Plasma Proteins and Immunoglobulins
51. Homeostasis and Thrombosis
52. The Red and White Blood Cells
53. Metabolism of Xenobiotics
54. The Human Genome Project
Biographical note

Robert K. Murray, MD, PhD,
Professor (Emeritus) of Biochemistry,
University of Toronto

David A. Bender, PhD,
Sub-Dean, University College Medical School,
Senior Lecturer in Biochemistry,
Department of Structural and Molecular Biology and
Division of Medical Education,University College London

Kathleen M. Botham, PhD, DSc,
Professor of Biochemistry, Royal Veterinary
College, University of London

Peter J. Kennelly, PhD,
Professor and Head, Department of Biochemistry,
Virginia Polytechnic Institute and State University,
Blacksburg, Virginia

Victor W. Rodwell, PhD,
Emeritus Professor of Biochemistry, Purdue University, West Lafayette,
Indiana

P. Anthony Weil, PhD,
Professor of Molecular Physiology and Biophysics,
Vanderbilt University School of Medicine,
Nashville, Tennessee

Co-Authors:

Daryl K. Granner, MD,
Emeritus Professor of Molecular Physiology and Biophysics
and Medicine,
Vanderbilt University, Nashville, Tennessee
Peter L. Gross MD, MSc, FRCP(C),
Associate Professor, Department of Medicine,
McMaster University

Frederick W. Keeley, PhD,
Associate Director and Senior Scientist, Research Institute,
Hospital for Sick Children, Toronto, and Professor,
Department of Biochemistry, University of Toronto

Peter A. Mayes, PhD, DSc,
Emeritus Professor of Veterinary Biochemistry, Royal Veterinary College, University of London

Margaret L. Rand, PhD,
Associate Senior Scientist, Hospital for Sick Children, Toronto,
and Professor, Departments of Laboratory Medicine and Pathobiology
and Department of Biochemistry, University of Toronto

download link
http://rapidshare.com/files/282803148/H_BC_28ed_lycan.rar

torrent link
http://freshwap.net/torrents/dl/2991880






Monday, February 15, 2010

Carbohydrate Chemistry

Introduction 

         Carbohydrates have the general molecular formula (CH2O)n, and thus were once thought to represent "hydrated carbon".

         All sugars are very soluble in water because of their many hydroxyl groups.

         They are called sugars because they are sweet in taste & crystalline in nature.

         Sugars are the most important source of energy for many cells.

Definition:

         Hydrates of carbon Cn(H2O)n

o       {exceptions:

         Acetic acid, C2H4O2 & lactic Acid, C3H6O3 Formaldehyde, CH2Oare not carbohydrates but

         Deoxy Ribose C6H12O5 is a carbohydrate

         carbohydrates are polyhydroxy aldehydes or ketones or compounds, which yields these on hydrolysis.

Classification:

         Hyaluronic acid

         Heparin

         Chondroitin SO4

         Keratan SO4

Carbohydrates

CARBOHYDRATES

Carbohydrates have the general molecular formula (CH2O)n, and thus were once thought to represent "hydrated carbon". All sugars are very soluble in water because of their many hydroxyl groups. They are called sugars because they are sweet in taste & crystalline in nature. Although not as concentrated a fuel as fats, sugars are the most important source of energy for many cells. Carbohydrates provide the bulk of the calories (4 kcal/gram)

1)Definition:

a)Hydrates of carbon Cn(H2O)n{exceptions: Acetic acid, C2H4O2 & lactic Acid, C3H6O3 Formaldehyde, CH2Oare not carbohydrates but Rahnose,C6H12O5 is.}

b)carbohydrates are polyhydroxy aldehydes or ketones or compounds, which yields these on hydrolysis.

2)Classification:

a)Monosaccharides:simple sugars that cannot be hydrolyzed to yield Carbohydrates

Name

Aldose

Ketose

Formula

Trioses

Glyceraldehyde

Dihydroxy acetone

C3H6O3

Tetroses

Erythrose

Erytrulose

C4H8O4

Pentoses

Ribose

Ribulose

C5H10O5

Hexoses

Glucose,Mannose, Galactose

Fructose

C6H12O6

Heptoses

Sedoheptulose

C7H14O7

Nonoses

Sialic acid or Neuramic acid

C9H18O9

b)Disaccharides: yields two molecules of Monosaccharides on hydrolysis

Name

Monosaccharides

Formula

Sources

Properties

Maltose

Glucose + Glucose

C3H6O3

Malt sugar, starch

Reducing

Lactose

Glucose + Galactose

C3H6O3

Milk Sugar

Reducing

Sucrose

Glucose + Fructose

C3H6O3

Cane sugar

Non-Reducing

Sucrose: prevalent in sugar cane and sugar beets, is composed of glucose and fructose through an a-(1,2)b-glycosidic bond.

Lactose: is found exclusively in the milk of mammals and consists of galactose and glucose in a b-(1,4) glycosidic bond.

Maltose: the major degradation product of starch, is composed of 2 glucose monomers in an a-(1,4) glycosidic bond.

c)Oligosaccharides: yields 3 to 10 Monosaccharides on Hydrolysis.

Maltotriose

Glucose + Glucose + Glucose

Raffinose

A trisaccharide that occurs in sugar beets and cotton seeds and cereals

Stachyose

A tetrasaccharide found in the tubers of the Chinese artichoke

d)Polisaccharides: yields more than 10 Monosaccharides on hydrolysis. They are of two types.

i)Homopolysaccharides (Homoglycans) Polymer of a single type of Monosaccharide.

Name

Monosacc.

Sources

Importance

Starch

Glucose

Potato,tapioca,rice,wheat,&grains

Plant source of Energy

Amylose

Glucose

Un branched starch similar to cellulose, containing alpha(1->4) linkages

Amylopectin

Glucose

Branched starch containing both 1,4 and 1,6 linkages.

Glycogen

Glucose

5% of liver mass> branched & compact

Animal storage of Glucose

Inulin

Fructose

various bulbs&tubers(onion,garlic)

Renal clearance studies.

Dextrins

Glucose

Product. ofstarch digestion

White dextrin is used in pharmaceutical preparations

Chitin

N-Ac-Glucosamine

Exoskeleton of Insects


 

ii)Hetroplysaccharides (Hetroglycans): polymer of two or more types of sugar residues

Agar

Glu +Gal + other

Obtained from Sea weeds

A colloidal extract of algae; used especially in culture media and as a gelling agent in foods (undigested by bacteria, dissolves at 100 oC and upon cooling sets into a Gel)

iii)MUCOPOLYSACCHARIDES (MPS) or GLYCOSAMINOGLYCANS (GAG)

Are Carbohydrates containing Uronic Acid & Amino Sugars.

Name

Functions

Hyaluronic acid

A viscous mucopolysaccharide found in the connective tissue space and the synovial fluid of movable joints and the humours of the eye; a cementing and protective substance

Heparin

A polysaccharide produced in basophils (especially in the lung and liver) and that inhibit the activity of thrombin in coagulation of the blood.

Chondroitin SO4

Inground substance of connective tissues (cartilage, tendon, bone)

Keratan SO4

In cornea & tendons.


 

e)Isomerism

i)D & L Isomerism

oThe configuration of the asymmetric carbon atom ( carbon atom whose 4 valencies are filled up by 4 different groups)farthest from the aldehydic or ketonic group

oIf –OH group is attached to the right then 'D' series & left then 'L' series.

ii)Pyranose - Furanose ring Structure

oWhen ring structure were suggested by Haworth (projections)

oFive member ring structure is calledFuranose and Six number as Pyranose

iii)Alpha & Beta Anomerism

oThe anomeric carbon (which originally was a part of the aldo or keto group) forms a hemiacetal or hemiketal in the ring structure generating a new asymmetric centre.

oIf hydroxyl gp (-OH) of the anomeric carbon is to the right then it is -form and if to the left it -form.

iv)Epimerism

oEpimers differ in the position of the Hydroxyl gp at only one of their assymetric C-atom.

oD-Glucose & D-Galactsoe are C-4 Epimers

oD-Glucose & D-Mannose are C-2 epimers

v)Enantiomers

oStereo isomers that are mirror images of each other

oEg: D-Glyceraldehyde & L-Glyceraldehyde.

vi)Mutarotation

oWhen D-Glucose is crystallized at RT and fresh solution is prepared, its specific rotation of polarized light is +112 o; but after few hrs it changes to +52.5 o. This change in rotation is called Mutarotation. This b'se D-Glucose has two anomers & forms. (-D-Glucose has specific rotation of +112 o and -D-Glucose has +19 o at equilibrium 1/3 is form and 2/3 is form to get a specific rotation of +52.5 o

3)Other Physiologically important carbohydrates.

Sugar

Present where

Importance&clinical Significance

D-Ribose

Nucleic acids

Nucleic acid co-enzymes.

D-Ribulose

Metabolic Product

Pentose-PO4 pathway intermediate.

D-Arabinose

Gum arabic, Plum & Cherry gum

Constituent of Glycoproteins

D-Xylose

Wood gums, GAG's

Constituent of Glycoproteins

D-Xylulose

Intermediate in Uronic acid pathway

Found in the Urine of patients with Essential pentosuria


4)CHEMISTRY

a)Reactions

i)Benedict's Reaction

Contains Na2CO3, Na-Citrte,CuSO4

In alkaline medium (Na2CO3), the copper remains as cupric hydroxide in presence of sodium citrate

5 ml Benedict's reagent+ 0.5 ml Urine boil for 2 minutes. If sugar present copper is reduced to produce green, yellow, orange, red precipitate depending on the concentration of the sugar.

SugarEnediolCu++(cupric)  CuSO 4(Reagent)

Sugar AcidCu+(cuprous) 2Cu(OH)[Yellow]Cu 2O [Red]

Chemical propertie

s

1

Reactions of aldehyde / keto group

  

Reactions

Reagents

Result

 

a.

Osazone formation

Sugar+Phenyl Hydrazine HCl+ Sodium Acetate (boil+cool)

Glucosazone-needle shapedLactosazone - Powder puff Maltosazone - Sunflower

b.

Reduction → Alcohol

Sodium amalgum / Hydrogen

Glucose - sorbitolFructose - Manitol

c.

Oxidation

  

I.

Mild (CHO → COOH)

Br2 H2O

Glucose - gluconic acid

ii.

Srong (CHO & CH2OH to COOH

HNO3

glucose - Glucosaccharic acid

iii.

Oxdn of CH2OH alone

Enzymatic (living cells only)

Glucose to glucuronic acid

d.

Tautomerisation( Inter conversions)

Weak alkaliBa (OH)2, Ca(OH)2

Glucose ↔ fructose ↔ Mannose

e.

Reduction Reactions

CuSO4, alkaline medium( cupric -> cuprous)

Benedicts, Fehlings & Barfoeds Test( Cuprous oxide- Red ppt)

2

Reactions of Alcoholic group

 

a.

Glycosides

1→4 &1→6 bond in glycosidic bonds in glycogen

  

I.

Cardiac

Derivatives of DIGITALIS,

Drugs in cardiac insufficiency

 

ii.

Oubin

strophanthus sp.

Sodium pump inhibitor (cardiac)

 

iii.

Phlorbizin

Root/bark of apple tree

block transport of sugar

 

iv.

Streptomycine

produced by the actinomycete Streptomyces griseus

Antibioticused to treat tuberculosis

 

b.

Acetilation/Ester formation

Alcohol + acids

phosphates ( glucose -1-phosphate)

 

c.

Dehydratin( Molisch, Selivanoffs, Bials tests)

Strong acids (Con.H2SO4)

Pentose→ Furfurals,Hexose→ Hydroxy methyl Furfural.

TESTS

 

Name of test

Substrate

Reagents

Result

1

Molisch's Test

Sugar

3d Alfa-Naphthol in Methanol +H2SO4 (side)

Violet color in the Junction.

2

Iodine test

Starch

Dil. Iodine

Deep Blue Colour

3

Benedict's

Reducing Sugar(0.5 ml/ 8 dps)

CuSO4+Na2CO3+NaCitrate(5 ml)

Cuprous Oxide (Red Precipitate)

4

Fehling's Test

Reducing Sugar

CuSO4+KOH+NaK Tartrate

Cuprous Oxide (Red Precipitate)

5

Barfoed's test

Monosaccharide.

CuAcetate+Dil.AceticAcid

(Red Precipitate) in < 5'

6

Bial's Test

Pentose

Orcinol Hydrochloride (Boil)

Green

7

SelivanoffsTest

Ketoses

Dil. Resorcinol in Dil.HCl (heat)

Cherry red

8

Tollent's Test

Reducing Sugar

Ammoniacal Silver nitrate

Metalic Siver Mirror

METABOLISM OF BILIRUBIN

METABOLISM OF BILIRUBIN

 
 

After 120 days of lifespan RBC's are broken down to release Hemoglobin

The hemoglobin then broken down to form Haem and Globin

            Globin being a protein is used as such or converted to amino acids and taken to amino acid pool

The Haem is then broken down to release a porphyrin compound and Iron

            The iron is taken to Iron pool and re utilized

The porphyrin ring opens and is converted to Biliveridin a green pigment

The biliveridin is immediately reduced to Bilirubin an yellow pigment

The formation of Bilirubin is taking place in the reticulo endothelial cells of  Spleen, Bone marrow and

 
 

The Bilirubin thus formed being water insoluble is then attached with the transport protein albumin and is then transported to liver through the blood. This albumin Bilirubin complex is known as the Unconjugated Bilirubin or Indirect Bilirubin or Free Bilirubin

 
 

Liver.

In liver there are three processes taking place

  1. Uptake of Bilirubin in to liver cells (hepatocytes)
  2. Conjugation of Bilirubin with Glucuronic acid
  3. Excretion of Conjugated Bilirubin in to Bile

The albumin is removed form the Bilirubin and is taken in to the  hepatocytes where they are conjugated.

            The Bilirubin in the hepatocyte is conjugated with two molecules of the UDP Glucuronic acid to form the Bilirubin Di Glucuronide and is known as the Conjugated Bilirubin or Direct Bilirubin.

            The conjugated Bilirubin being water soluble is excreted in to the bile and it reaches the small intestine

Small Intestine:

In small intestine the glucuronides are removed and the Bilirubin is converted to urobilinogen by the intestinal bacteria.

A part of the urobilinogen is reabsorbed back in to the liver through the portal circulation and reaches the liver and thus in to the general circulation. Then it is excreted in to the urine via kidneys. The urobilinogen in urine is the cause of yellow coloration to urine and when it is exposed to oxygen is converted to dark Urobilin.

The remaining urobilinogen in the small intestine is excreted in to the feces as Stercobilinogen. This Stercobilinogen in the stool is the cause of yellow coloration to the stool and when it is exposed to the oxygen it is converted to dark colored stercobilin.

Adsorption Chromatography

INTRODUCTION

  • Adsorption chromatography is probably one of the oldest types of chromatography around.
  • It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase.
  • The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.

PRINCIPLE

  • Classic form of Chromatography by Tswett  

Principle

  • Based on the principle that certain solid materials, collectively known as adsorbents have the ability to hold molecules at their surface.
  • This adsorbing process which involves weak non-ionic attractive forces of van der Waal's and hydrogen bond occurs at specific adsorption site.
  • These sites have the ability to discriminate between the molecules and the adsorption of the molecule depends on the strength of the interaction.
  • As elutes continuously pass down the column, difference in their binding eventually lead to the separation for the analytes.
  • Binding depends on the functional groups present in the molecules
    • Hydroxyl, aromatic groups - increase interaction with the binding surface
    • Aliphatic groups - decrease interaction with the binding surface

STATIONARY PHSAE / ADSORBENT

Silica

  • with Silanol groups (Si-OH) groups on its surface
  • Are slightly acidic in nature
  • Can interact with polar functional group of the analyte or eluent.
  • The topology (arrangement of Si-OH explains their differential separation
  • Silica is acidic - good for the separation of basic substances
  • Available for both LPLC & HPLC

Alumina

  • Available for both LPLC & HPLC
  • Alumina is basic - good for the separation of acidic substances

Carbon

  • Available for both LPLC & HPLC

MOBILE PHASE ELUENT

  • Depends on k' (partition ratio / capacity ratio) of analyte. (which is the time spent by analyte in the stationary phase relative to the time spend in the mobile phase)
  • Eluent with polarity comparable to the most polar compound in the analyte mixture is selected as mobile phase.
    • Alcohol is selected for analytes with hydroxyl (-OH) groups
    • Acetone/esters is selected for analytes with carbonyl groups (=C=O) groups
    • Hexane / heptane /toluene (hydrocarbons) is selected for non polar analytes
    • Mixture of solvents also can be used in gradient elution.
  • Presence of small amounts of water in the MP is beneficial when silica is the stationary phase – which selectively block more active Silanol groups leaving a more selective population of weaker binding sites.

MODES/ APPLICATION -USES

Modes

  • Column
  • Thin layer

Application/USES

  • Most commonly used to separate non ionic water insoluble substances
    • Triglycerides
    • PTH
    • Aminoacids
    • Vitamins
    • Many drugs

TWO MAJOR TYPES

  • Hydroxylapatite Chromatography
  • Hydrophobic interaction  Chromatography

HYDROXY(L)APATITE CHROMATOGRAPHY

  • Also known as hydroxyapatite chromatography
  • Adsorbent used
    • Crystalline  hydroxyapatite (Ca10(PO4)6(OH)2)
  • Mechanism
    • Involves both Ca & PO4 ions on the surface
    • Involve dipole-dipole interaction
    • Involve electrostatic interaction

APPLICATION/ USES OF HC

  • Used to separate single stranded DNA from double stranded DNA
  • Both single stranded and double stranded binds at low phosphate buffer concentration
  • At increased buffer concentration single stranded DNA is selectively desorbed
  • Further increase in buffer concentration desorbs double stranded DNA also
  • Used of Cot Analysis
  • So DNA can be separated from mixture of RNA and protein of cell Extracts.

COMMERCIAL

  • Suitable for both LPLC & HPLC
  • Crystalline or spheroidal hydroxy apatite is bonded to the agarose matrix
  • Adsorption capacity is maximum around the neutral pH using 20 mM phosphate buffer
  • Elution is done at 500 nM

HYDROPHOBIC INTERACTION CHROMATOGRAPHY

  • Used to purify protein exploiting their surface hydrophobicity (non polar amino acids)
  • In aqous solution these hydrophobic regions of proteins are covered with an ordered film of water molecules that effectivel mast these hydrophobic groups
  • Exposed by the addition of salt ions
  • Then these hydrophobic regions interact together and is the basis of salting out by the addition of ammonium sulphate
  • If hydrophobic groups are attached to a suitable matrix the hydrophobic groups on the proteins will interact with them on the matrix to facilitate a protein-matrix attraction
  • Most commonly used stationary phases are
    • Alkyl (hexyl , Octyl)
    • Phenyl groups
    • Attached to Agarose matrix

Commercial

LPLC(HIC)

  • Phenyl Sepharose , Phenyl SPW

HPLC (HIC)

  • Biogel TSK phenyl, Spherogel TSK Phenyl
  • Ammonium sulphate is present in the sample since it fractionated before analysis with it which help to expose the hydrophobic centers.

Elution process.

  • Gradually decrease the ionic strength
  • Increase the pH
  • Selective displacement by displaces that has s stronger affinity for the stationary phase than has the protein
    • Non ionic detergents like
      • Tween 20
      • Trinton –X100
    • Aliphatic Alcohols like
      • Butanol
      • Ethylene Glycol
    • Aliphatic Amines
      • Butylamine
  • Some of these elution condition may cause protein denaturation
  • Protein purified -->Aldolase, Transferrin, Cytochrom c, thyroglobulin etc.