NMR SPECTROSCOPY IN BIOLOGY

CONTENTS

• Introduction

• Basic principles in NMR spectroscopy

• Instrumentation

• Interpretation of NMR spectra

• Applications in the field of biology

Introduction

introduction

History• First described and measured by Isidor Rabi in

1938.

• In 1946, Felix Bloch & Edward Mills Purcell refined the technique for use on liquids & solids for which they shared the Nobel Prize in physics in 1952.

• The observation of resonance frequencies of a molecule allows a user to discover structural information about a molecule.

Contd…Contd…

Basic Principles in NMR Spectroscopy

basic principle in NMR spectroscopy

Magnetic phenomena• Atomic nuclei – tiny magnets

• In external field – align or oppose

Number of nucleons • Must be odd for residual magnetism

• For e.g. Hydrogen exhibits, Deuterium does not

Contd…Contd…

Resonance condition• Transition from low to high energy state

occurs

• hע = gßH

• Either a constant magnetic field is applied and appropriate region of RF is swept

• Or a constant frequency is employed & the external magnetic field is swept

Contd…Contd…

basic principle in NMR spectroscopy

Chemical shift• Arises from applied field inducing secondary

fields at the proton by interacting with adjacent bonding electrons

• Is measured relative to an internal standard – TMS

Spin – spin splitting• Seen in high resolution spectroscopy

basic principle in NMR spectroscopy

Contd…Contd…

E.g. of ethanol

a) Low resolution spectrum b) High resolution spectrum

Contd…Contd…

Instrumentation

Diagram of NMR unit

Contd…Contd…

instrumentation

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Interpretation of NMR Spectrum

Number of main signal = number of equivalent protons in unknown compound

Chemical shift indicates the type of H atoms. E.g. methylene, methyl groups, etc.

Spin spin splitting ---- arrangement of groups in the molecule.

Area of peaks ---- no. of H nuclei present in each group. For e.g. relative areas of methyl peaks in propane would be 6:2. In butane it would be 6:4

E.g. of Ethanol

Application in the field of Biology

Application in the field of Biology

Molecular structure determinationMost work done in solutions.

Drug metabolism studies & mechanism of drug action.

Low resolution NMR on lac repressor headpiece and BPTI.

High resolution NMR spectrum on structure of BDS-I, C3a & C5a inflammatory proteins, plastocyanin, thioredoxin, epidermal growth factor & interleukines

Solid state NMR --- Alzheimer’s B-Amyloid peptide.Contd…Contd…

E.g. of Ibuprofen

Molecular behavior in solutions

Results of studies of protein folding are exemplified by ribonuclease A, cytochrome c, barnase, lysozyme, ubiquitin and BPTI.

Enzyme kinetics, e.g. : chymotrypsin, trypsin, papain, pepsin, ATPase, ribonuclease, pyruvate kinase, etc.

Contd…Contd…

Phosphate metabolism

31P isotope exhibits nuclear resonance

Changing concentration of AMP, ADP & ATP in living cells

Intracellular & extracellular Pi concentrations

Contd…Contd…

Nucleic acidsStructural studies of DNA & RNA

Drug interactions with DNA

Interactions between binding proteins & DNA

Interaction between protein & lipid bilayers in membranes have been observed.

Contd…Contd…

Magnetic Resonance Imaging (MRI)

Resonance of 1H is considered.

Proton is more sensitive.

Present in abundance.

Contd…Contd…

a)Human brain

Imaging of posterior fossae, brain stem, spinal cord

Detection of demyelinated lesions, hemorrhages.

Differences in water content in white and gray matter in brain & between normal tissues and tumors.

Contd…Contd…

b)Abdomen

Metabolic liver disease.

In adipose tissue --- proton signals from lipids is measurable & hence the fatty deposits can be viewed.

Contd…Contd…

Advantages of MRI

Eliminates risk of X-radiation.

Excellent and contrast resolution.

Detecting disease at earlier stages.

Advantages of NMR spectroscopy

• A considerable amount of information can be obtained from a single analysis

• The technique is non destructive

• Replicate analysis may be performed

• Precious samples may be recovered

References

Principles and techniques in Biochemistry & Molecular biology, by Keith Wilson & Walker, 8th edition.

Nuclear Chemistry by R. K. Dave Biochemistry with clinical correlation by

Thomas M. Delvin Websites:

http://biologyreference.com http://chemnmr.colorado.edu/microinfo