DETECTION OF PEPTIDE HORMONES

BY: Reddy Sailaja M (MSIWM030)

HORMONES

A hormone is a signaling molecule secreted by endocrine glands in response to physiological stimuli in multi cellular organisms. These hormones circulate in blood and reach its destination to exert specific function. Hormones help maintain physiological and behavioral functions in the organisms.

Hormones are classified into three main classes:

• Steriod hormones – Lipid soluble and move across plasma membrane of the targeted cells.
• Peptide hormones – Water soluble and act through cell surface receptors present on the targeted cells.
• Aminoacid derivatives

Table 1: Three classes of hormones

Peptide hormones

Peptide hormones are made up of small amino acid chains called, peptides. Peptide hormones are synthesized in the cells from amino  acids based on mRNA sequence that is derived from DNA template within the nucleus.

Peptide hormones can’t navigate across plasma membrane of the cell. Hence, they exert their function by binding to the receptor present on the cell surface of the target cell that in turn trigger signal transduction and cellular response. Some peptide hormones like parathyroid hormone-related protein, angiotensin II etc interact with intracellular components within cytoplasm or nucleus by an intracrine mode of interaction.

Some of the examples of peptide hormones are as follows:

• Adrenocorticotrophic hormone
• Thyroid stimulating hormone
• Vasopressin
• Angiotensin II
• Antrial natriuretic peptide
• Calcitonin
• Follicle stimulating hormone
• Insulin
• Growth hormone
• Parathyroid hormone
• Prolactin

Table 2: Main peptide hormones – details and functions

Detection of peptide hormones

As peptide hormones circulate in blood to reach their destination, serum generally acts as the source of detection and measurement.

The following are the main detection methods used to detection peptide hormones:

1. Sandwich ELISA technique:
2. In sandwich ELISA method, two antibodies are used to detect hormone of interest. One of the antibodies is attached to the solid support on the micro titer plate, called as capture antibody.
3. Second antibody labeled with a signal molecule (enzyme or radioisotope or chemilumiscent) acts as detector.
4. When the analyte containing mixture is loaded onto the micro titer plate, capture antibodies bind to the analyte via epitope that is present on the analyte surface and catch hold of analytes.
5. When the detector antibodies are added to the plate, they bind to different location on the analyte.
6. In the enzyme based reaction, when substrate is added, it reacts with enzyme that is attached to the detector body and shows response in the form of color change.

Figure 1: Sandwich ELISA to detect analytes in the blood

• Radioimmunoassay (RIA):
• RIA is an in vitro detection technique that detects and measures antigens (like hormones and other foreign substances) in the blood. RIA technique is discovered by Berson and Yalow in 1960 to analyze insulin levels in blood.
• RIA method is based on the radioactivity measurement associated with antigen-antibody interactions in the reaction.
• A known antigen that is radiolabelled is incubated with antibody at known concentrations.
• When the analyte containing solution is added to the labelled antigen-antibody mixture, antigen of interest replaces labelled antigen and bind to the antibody.
• More the antigen of interest present in the solution, more labelled antigen will be displaced and replaced with antigen of interest.

Figure 2: Radioimmunoassay

• Enzyme multiplied Immunoassay Technique (EMIT):
• EMIT is more easy and equivalent detection method, both qualitatively and quantitatively to measure wide-range of analytes from the serum.
• EMIT is based on the principle that the amount of analyte present in the solution is directly proportional to the inhibition of enzyme-substrate reaction complex.
• In this technique, initially a known analyte is labelled with an enzyme and antibody specific to drug is allowed to bind drug-enzyme complex. This results in inhibition of enzyme activity.
• When the solution containing analyte is added to the above mixture, the analyte releases the antibody from the drug-enzyme complex, thereby increasing enzymatic activity.
• Therefore, enzyme activity is proportional to the analyte present in the sample added and is measured by absorbance value changes of the enzyme.

Figure 3: Enzyme multiplied Immunoassay Technique

• Immunoradiometric assay (IRMA)
• IRMA utilizes radiolabeled antibodies to detect analytes of interest.
• In this technique, antibody is directly labeled with radioisotopes rather than using two antibodies as in other immune assays.

• When an analyte containing solution is added to micro titer plateradiolabeled antibodies bind to the specific epitopes of the anlytes and forms the antigen-antibody complex.

• I125 and I131 radioisotopes used in general for this assay.
• Unbound radiolabeled antibodies are removed from the plate by second wash during the process.

Figure 4: Immunoradiometric assay

The other peptide hormone detection methods are listed below:

• Ultrafiltration
• Chromatography
• Time resolved fluorescence
• Mass spectrometry
• Two site immunometric technique

PROTIENS STRUCTURE, CLASSIFICATION AND FUNCTION

                   BY: Sreelakshmi S Nair

Proteins:

Proteins are macromolecules obtained from the one or more amino acids chain linked by peptide bonds. They are the natural polymers of amino acids. It contains nitrogen, carbon, hydrogen and oxygen. They act as a biological catalyst form structural parts of different organisms, participate in different cell reactions.

CLASSIFICATIONS OF PROTIENS

Proteins are classified on the basis of

• Structure of Protein
• Composition of Protein
• Functions of Protein

Based on the structure of Protein

• Fibrous Protein

They are linear in shape. Usually they do not have tertiary structure. They are physically strong and are insoluble in water. They perform the structural functions in the cell. Examples are: Keratin, Collagen, and Myosin.

• Globular Protein

They are spherical or globular in shape.Teritary structure is the most functional structure. Physically they are soft while comparing to fibrous proteins. They are readily soluble in water. Most of the proteins present in the cell belongs to globular protein. It forms enzymes, antibodies and some hormones. Examples are Insulin, haemoglobin, DNA polymerase and RNA polymerase.

Based on the composition of Protein

• Simple proteins

They are composed of only amino acids. They may be fibrous or globular. They are generally simple in structure. Examples are Collagen, Myosin, Insulin

• Conjugated Proteins

They are complex proteins which contains one or more amino acid components. The non-protein parts are called prosthetic group. Prosthetic group may contain metals, ions, carbohydrates, lipids, nucleic acid. Conjugated proteins are generally water soluble and globular in structure. Most of the enzymes are conjugated proteins.

Based on the Function

• Structural Proteins

Most of them are fibrous proteins. Components in connective tissue, bone, tendons, cartilage, skin, feathers, nail, hairs and horn are made of structural proteins.

• Enzymes

They are the biological catalyst. Mostly globular conjugated proteins. Examples are DNA polymerase, Nitrogenase, and Lipase.

• Hormones

They include proteinaceous hormone in the cells. Examples are Insulin, ACH

• Respiratory Pigments

They are coloured proteins. All of them are conjugated proteins. Examples are haemoglobin, Mycoglobin.

• Transport Proteins

They transport the materials in the cell. They form channels in the plasma membrane. They also form a component in blood and lymph in animals.

STRUCTURE OF PROTEIN

There are four levels

1. Primary Structure
2. Secondary structure
3. Tertiary Structure
4. Quaternary Structure

1. Primary Structure

They give details about the amino acid sequence of a protein. It tells about the number of amino acid residues in the protein and also about the sequence of amino acids. It is stabilized by peptide bonds. Each component of an amino acid is called residue or moiety. It starts from the amino terminal (N) end and ends in the carboxyl terminal(C) end.

• Secondary structure

It is formed by hydrogen bond between backbones atoms. Three most important secondary structure in protein are:

• α-helix
• β- plates
• β-Turns

α -Helix

It is the most common secondary structure which repeat every 5.4It is the simplest arrangement of a polypeptide chain which was proposed by Puling and Corey in 1954.It is so common because it makes optimal use of internal hydrogen bond. The interactions between the amino acid chains can stabilize or destabilize the α-helix.

                    β- Plates

It is an extended form of a polypeptide chain.Polypetide backbone forms a zigzag structure. Similar to α-Helix structure is stabilized by hydrogen bond. The R-groups of adjacent amino acids protrude from the zigzag structure to the opposite direction forming an alternative pattern. It can be arranged in either parallel or anti-parallel direction.

                    β-Turns

         It’s a very common in proteins where peptide make a reverse direction. It forms a 180 degree turn involving four amino acids. The carbonyl oxygen of the first residue forms a hydrogen bond with the amino group hydrogen in the fourth amino acid in the turn. Glycine and proline allows the β-Turns frequently.

3. Tertiary Structure

                   The tertiary structure will have a single polypeptide backbone consisting of one or more    secondary structures. It can be defined by atomic coordinates. It is stabilized with the help of both covalent and non-covalent bond.

4. Quaternary Structure

Proteins which have more than one polypeptide subunit and which do not have a permanent (covalent) interaction between the subunits (like disulphide bond) are classified under quaternary structure. Bonds stabilizing quaternary structure includes hydrogen bonds, hydrophilic interactions, hydrophobic interactions, van der Waals interactions. A protein with a single subunit cannot have a quaternary structure.

Functions of Protein

• Boosts Immune System
•  Provides Structure
• Maintains pH
• Transports and stores nutrients