Food Science: Proteins and Cell Structure

• Fundamentals of Science covers Level 6 Certificate in Food Science and Technology.
• Megan Kelly from innopharmalabs.com can provide more information.

Module Content

• Biology covers cell structure and function, proteins and analysis, lipids, carbohydrates, nutrition, enzymes, quantitative analysis, and weights, measures, and preparation.
• Chemistry includes the periodic table, chemical bonds, acids and bases, and elemental analysis.

Session 1 Agenda

• Cell Structure and Function followed by a 10-minute break.
• Proteins are discussed and followed by a 15-minute break.
• Protein Analysis.

Session 1 Learning Objectives

• Define the structure and function of proteins, including amino acids.
• Describe the protein synthesis process.
• Detail the impact of denaturation on proteins.
• Describe methods of protein analysis.

Function of Proteins

• Proteins are the body's building blocks and categorized as structural or functional.
• Structural proteins include collagen fibers found in hair, wool, and silk.
• Functional proteins include enzymes which break down sugars and DNA.
• Receptors are functional proteins where drugs bind, affecting cell fate.
• Movement is aided by functional proteins like muscles.
• Functional proteins include defense proteins like antibodies.
• Hemoglobin is a functional protein that transports oxygen in the blood.

Protein Composition

• Proteins consist of 20 amino acids which are their building blocks.
• All amino acids contain carbon, hydrogen, oxygen, nitrogen, and most contain sulfur.
• Some amino acids include iron, zinc, or copper.
• Proteins typically have a high molecular weight (MW).
• The structure of a protein can be broken down by hydrolysis.
• A broken protein results in loss of function.

Amino Acid Components

• Twenty different amino acids each contain 1 carboxyl group and 1 amino group.
• The R group/side chain makes each amino acid unique.

Protein Structure

• Proteins are composed of amino acids linked by covalent bonds, resulting in long, unbranched chains.
• Covalent bonds forming these chains are known as peptide bonds.
• Once a protein is made, it folds into a 3D structure, which is essential for bioactivity.
• Mutations may cause a loss or change in activity.

Peptide Bond Formation

• A peptide bond is the linkage between two amino acids.
• Peptide bonds are formed by condensation reactions.

Protein Classification by Polarity

• Proteins can be classified as hydrophilic or hydrophobic.

Polypeptide Chains

• Peptide refers to a chain of amino acids linked together by peptide bonds.
• Polypeptides usually refer to long peptide chains.
• Proteins consist of one or more polypeptides containing more than 50 amino acids.

Protein Structure Types

• Proteins have primary, secondary, tertiary, or quaternary structures.

Primary Structure

• The primary amino acid sequence of a given protein is unique and genetically determined.
• Mutations to a gene alter the amino acid sequence; for example, cystic fibrosis.

Secondary Structure

• The chain of amino acids twists into spirals: alpha helixes.
• Beta pleated sheet shapes.
• These formations are due to hydrogen bonds.

Tertiary Structure

• The overall three-dimensional structure of a polypeptide chain.
• Tertiary structure is primarily due to interactions between the R groups of the amino acids.

Quaternary Structure

• Two or more tertiary structures bind together to form a larger overall structure.
• Hemoglobin requires a quaternary structure.
• Hemoglobin carries oxygen in the body and also needs iron.

Protein Digestion

• Proteins are broken down by enzymes in the digestive system.
• Polypeptide chains are broken down into amino acids which enter the bloodstream and cells.
• Amino acids are the building blocks for other cellular proteins.

Protein Denaturation

• Denaturation changes or breaks protein structure, leading to loss of activity.
• Denatured proteins have different properties compared with native proteins.

Causes of Protein Denaturation

• High temperatures, extremes in pH, and vigorous mixing (whisking).
• Denatured proteins lose their shape and become inactive.
• Proteins cannot go back to their original shape upon denaturation.

Handling Proteins

• To avoid denaturation proteins should:
  • Avoid high temperatures.
  • Refrigerate.
  • Avoid freeze-thaw cycles.
  • Keep a neutral pH is best.
  • Avoid vigorous mixing.

Revision Questions on Proteins

• Features of proteins.
• Four structural types of proteins.
• The two types of secondary protein folds.
• Example of a quaternary protein.
• What happens as part of protein digestion?
• How can proteins be denatured?
• Considerations handling proteins in a lab?

Protein Analysis: Thin Layer Chromatography (TLC)

• Amino acids can be identified using thin layer chromatography (TLC).

Protein Analysis: Spectrophotometry

• A way to measure protein using spectrophotometers.
• The amount of light proteins absorb is measured and related to a standard curve with known protein concentrations.
• Linear regression & equation of the line can then be used.
• Various assays measure the amount of protein in a solution.

Protein Measurement at 280 nm

• This depends on the presence of three aromatic amino acid residues.
• Tryptophan, phenylalanine, and tyrosine can absorb ultraviolet light strongly at 280 nm.
• The absorbance of protein solutions at 280 nm can be used to determine protein concentration.
• Advantages of the method are its simplicity, non-destructive nature, and no special reagents are required.
• Nucleic acids also absorb strongly at 280 nm, which can be the major disadvantage and interfere.
• Commonly used method where the protein is required for further analysis.

Protein Determination

• All proteins have aromatic amino acids.
• All proteins absorb light at 280 nm.
• Requires an instrument, the UV-Visible spectrophotometer, and quartz cuvettes for sample holders.

Multi-point Calibration

• A series of 5-6 standards which are then plated on Abs at 280 nm vs. conc.
• Linear regression analysis is then undertaken.
• Equation of line then used to quantify protein.

Beer-Lambert Law

• For a given material sample path length and concentration of the sample are directly proportional to the absorbance of the light.

Multi-Point Calibration Method

• Set up all relevant equipment for multi-point calibration.
• Prepare a series of dilution standards at regular intervals (5-6 samples).
• Run the standards at 280nm within the UV spec.
• Plot the data.
• Apply a calibration curve.
• Fit the data to linear regression (ensure as close to 1 as possible).
• Get the equation of the line y=mX + C (eg X= (y-c)/m).

Biuret Method

• Cupric ions (Cu2+) interact with peptide bonds under alkaline conditions.
• Violet-purplish colour is produced.
• A commercially available reagent is mixed with a protein solution and allowed to stand for 15-30 minutes before absorbance is read at 540 nm.
• Advantage: there's no interference from materials that adsorb light at this wavelength.
• Disadvantage: relatively poor sensitivity compared to other UV-visible methods (mg/ml).

Lowry Method

• Combines the biuret reagent with another reagent, the Folin-Ciocalteau phenol reagent.
• Reacts with tyrosine and tryptophan residues in proteins.
• Produces a blue colour is read at either 500 or 750 nm.
• High Protein Concentration = 500 nm
• Low protein Concentration = 750 nm.
• Advantage: more sensitive to low concentrations of proteins than the biuret method (ug/ml).
• Read absorbance of unknown sample against known calibration curve to determine concentration.

Summary of Protein Analysis

• Detect using qualitative analysis and quantify using quantitative analysis.
• Use of Spectroscopy at 280 nm – protein is not damage & can be used (recovered) for other experiments.
• Use of Spectroscopy with Biuret or Lowry reagent means the protein is measured and then disposed of (cannot be recovered).

Revision Questions on Methods of Protein Analysis

• Three types of protein analysis that can be used.
• Protein analysis that does not use a reagent.
• What is the advantage of Lowry over Biuret?
• How to use a calibration curve to determine the concentration of an unknown protein?

Explore the fundamentals of proteins, cell structure and function. This lesson covers the roles of proteins as the body's building blocks and describes protein synthesis. It also details the impact of denaturation on proteins and various methods of protein analysis.