Document (7).pdf - Biochemistry Lab Manual PDF

BAPNA (N-Benzoyl-DL-arginine-p-nitroanilide) is a synthetic substrate used to measure proteolytic enzyme activity. Proteases like trypsin cleave BAPNA, releasing a yellow color (p-nitroaniline), which indicates enzyme activity. The intensity of the color reflects the level of proteolysis. Factors t...

BAPNA (N-Benzoyl-DL-arginine-p-nitroanilide) is a synthetic substrate used to measure proteolytic enzyme activity. Proteases like trypsin cleave BAPNA, releasing a yellow color (p-nitroaniline), which indicates enzyme activity. The intensity of the color reflects the level of proteolysis. Factors that Denature Proteins (according to the file): 1. Heat: High temperatures disrupt protein structures. 2. pH Changes: Extreme acid or alkaline conditions cause proteins to lose their natural structure. 3. Alcohol: Can alter protein folding by breaking hydrogen bonds. 4. Heavy Metals: Ions like mercury or lead bind to proteins and disrupt their structure. 5. Mechanical Agitation: Stirring or shaking can cause proteins to denature. Whipped Cream After Lipase Hydrolysis (according to the file): Texture Change: After hydrolysis by lipase, whipped cream becomes watery due to the breakdown of fats into glycerol and fatty acids. Release of Fatty Acids (Rancid Taste) (according to the file): Rancid Taste: The breakdown of triglycerides by lipase releases fatty acids, which gives the rancid taste observed in the cream due to the incomplete oxidation of fats  Identifications of Compounds Based on Structures: 1. TNP (Tri-nitrophenol):  Structure: A highly acidic phenol with three nitro groups attached to the benzene ring. Identification: TNP is used to test for certain compounds, especially in the presence of reducing agents. Positive Result: Often identified in chemical reactions involving reducing sugars or other phenolic compounds. 2. Uric Acid: Structure: A purine derivative (C5H4N4O3) that crystallizes in the form of yellowish-brown crystals. Identification: High levels of uric acid in urine are associated with gout. Test: Murexide Test results in a bluish-violet color, indicating uric acid or its derivatives. 3. Creatinine: Structure: A nitrogenous waste product (C4H7N3O) resulting from muscle metabolism. Identification: Creatinine levels in urine can indicate kidney function. Test: Excreted in a consistent amount by healthy individuals, typically 1.2-1.7 g/h. 4. Indican: Structure: A product of the breakdown of tryptophan in the intestine, related to intestinal putrefaction. Identification: Obermeyer’s Test gives a greenish-blue result indicating indican’s presence. 1. Abnormal Constituents in Urine: Abnormal Constituents refer to substances that are not typically present in healthy urine or are present in abnormal amounts. These include: Proteins (e.g., albumin, globulin) Glucose Ketone bodies  Blood cells (red or white) Bacteria Crystals (e.g., urates, phosphates) 2. Pathological Constituents (to memorize): Proteins: Presence of proteins (proteinuria) suggests kidney damage, such as in nephritis or kidney failure. Glucose: Presence of glucose (glycosuria) is often linked to diabetes mellitus or excessive carbohydrate intake. Ketone Bodies: These are produced during fat metabolism, typically seen in diabetes, starvation, or increased fat oxidation. Bence-Jones Proteins: Indicative of multiple myeloma or other blood plasma disorders. These proteins precipitate at low temperatures and dissolve at higher temperatures. 3. Indicators of Liver Problems: Bilirubin: A breakdown product of hemoglobin, elevated levels in urine may indicate liver disease (e.g., jaundice). Urobilinogen: A decrease or increase in urobilinogen in urine can indicate liver dysfunction or hemolytic disease. 4. Key Indicators for Diabetes: Bence-Jones Proteins: May be present in urine, signaling possible multiple myeloma. Ketone Bodies: These indicate ketoacidosis, a severe condition of uncontrolled diabetes mellitus.  Urea: Urea is a nitrogenous waste produced from protein metabolism and is excreted in urine. Its structure is: Formula: CO(NH₂)₂ Structure: A carbonyl group (C=O) attached to two amine groups (NH₂). 5. Interpretation of Test Results: Dipstick Test: Commonly used to test for: pH (range: 4.5 to 8.2) Glucose (positive result: indicates diabetes or high sugar intake) Proteins (positive result: suggests kidney damage) Ketones (positive result: indicates diabetes, starvation, or other metabolic disturbances) Microscopic Examination: Used to identify cells, bacteria, and crystals in urine, which can help diagnose conditions like urinary tract infections (UTIs) or kidney stones. 6. Situational Problems: In situational problems, interpret abnormal test results based on known conditions: Example: If a patient shows high glucose and high ketones in their urine, this could suggest diabetic ketoacidosis. Example: Proteinuria in a patient with high blood pressure could suggest kidney damage or nephropathy. Biuret Test: For proteins, indicating kidney issues. Benedict’s Test: For glucose, indicating diabetes.  Acetone Test (Legal’s Test): For ketone bodies, indicating fat breakdown in diabetes or starvation. Fehling’s Test: For reducing sugars (like maltose). Nitric Acid Test: For protein, used to detect kidney issues or albuminuria.