SDS Used? Yes (in sample, gel, buffer) No Reducing Agent? Yes (to break disulfide bonds) No Charge Uniformly negative (SDS-bound) Intrinsic charge of protein Protein Activity Lost Retained (can be assayed) Applications MW determination, purity, quantification Protein interactions, enzyme activity, isoforms

7. Blood Glucose Estimation / Enzyme assay (15 Marks)

Blood Glucose Estimation (Enzymatic Method)

Principle: Enzymatic methods are highly specific and sensitive for measuring glucose in blood. The most common method uses glucose oxidase and peroxidase.
Enzymes Involved:
- Glucose Oxidase (GOD): Catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide ($H_2O_2$).
- Peroxidase (POD): Catalyzes the oxidation of a chromogenic substrate (e.g., o-toluidine, 4-aminophenazone, phenol) by $H_2O_2$ to produce a colored product.
Reaction Steps:
1. Glucose + $O_2$ + $H_2O$ $\xrightarrow{\text{Glucose Oxidase}}$ Gluconic Acid + $H_2O_2$
2. $H_2O_2$ + Chromogen $\xrightarrow{\text{Peroxidase}}$ Colored Product + $H_2O$
Measurement: The intensity of the colored product is directly proportional to the glucose concentration and is measured spectrophotometrically at a specific wavelength.
Procedure Overview:
1. Collect blood sample.
2. Prepare plasma or serum (or use whole blood for point-of-care devices).
3. Add a known volume of sample to a reagent containing GOD, POD, and the chromogen.
4. Incubate for a set time at a specific temperature.
5. Measure absorbance of the colored product using a spectrophotometer.
6. Compare absorbance to a standard curve of known glucose concentrations.
Clinical Significance: Diagnosis and monitoring of diabetes mellitus, hypoglycemia, hyperglycemia.

General Principles of Enzyme Assay

Definition: A method to measure the activity of an enzyme by quantifying the rate at which it converts its substrate into product.
Reaction Rate: Enzyme activity is typically expressed as the amount of product formed or substrate consumed per unit time (e.g., $\mu mol/min$).
Factors Affecting Enzyme Activity:
- Substrate concentration.
- Enzyme concentration.
- pH.
- Temperature.
- Presence of activators or inhibitors.
Types of Enzyme Assays:
- Continuous Assays (Kinetic Assays): Measure the change in substrate or product concentration over time, providing a reaction rate.
  - Often involve a direct spectrophotometric measurement if the substrate or product absorbs light, or a coupled reaction that produces a detectable signal.
  - Example: Measuring NADH oxidation at 340 nm.
- Discontinuous Assays (Endpoint Assays): The reaction is stopped after a fixed time, and the amount of product formed or substrate consumed is measured.
  - Suitable when the product or substrate is stable after the reaction i