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*BCH 202 – General Biochemistry II | 1-Page Revision Sheet*
*1. Centrifugation*
*Definition*: Separation of particles in a mixture using centrifugal force based on size/density.
*RCF* = 1.118 × 10⁻⁵ × r × (RPM)²
_r = radius in cm, RPM = revolutions per minute_
*Types*:
– *Preparative*: Isolate cells, organelles, proteins for use.
– _Differential_: Spin at increasing speeds. Large/dense particles pellet first.
– _Density gradient_:
– _Rate-zonal_: Separate by size/mass. Particles form bands as they sediment.
– _Isopycnic_: Separate by density. Particles settle where gradient density = particle density.
– *Analytical*: Study purified molecules. Measures sedimentation velocity, MW, purity.
*Rotors*:
– *Fixed angle*: 14-40° to vertical. Fast pelleting, used in differential.
– *Swinging bucket*: Swing horizontal on acceleration. Better for gradients.
– *Vertical*: Tubes vertical. Short separation distance, used for isopycnic.
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*2. Cell Fractionation*
*Goal*: Isolate organelles from cells.
*Steps*:
1. *Homogenize* tissue in isotonic sucrose buffer to prevent organelle swelling/bursting.
2. *Differential centrifugation*:
– 600g → nuclei, debris
– 10,000g → mitochondria, lysosomes
– 100,000g → microsomes, ribosomes
3. *Isopycnic centrifugation*: Layer homogenate on sucrose gradient. Collect organelles at their buoyant density.
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*3. Water & Electrolyte Balance*
*Total body water*: Men 55-70%, Women 45-60% of body weight.
*70 kg man*: 42L total = 28L ICF + 14L ECF. ECF = 10.5L interstitial + 3.5L plasma.
*Water intake*: Drinking + food + metabolic water ∼300-350 mL/day.
*Water loss*: Urine, skin, lungs, feces. Controlled by thirst center in hypothalamus.
*Key electrolytes*:
– *ECF*: Na+, Cl-, HCO₃⁻
– *ICF*: K+, HPO₄²⁻, proteins, organic acids
Maintained by *Na⁺/K⁺ pump* and hormones: aldosterone [Na⁺ retention], ADH [water retention], renin-angiotensin, ANF [Na⁺ excretion].
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*4. Acid-Base & pH*
*Normal blood pH*: 7.35-7.45. Life range: 6.8-7.8.
*pH* = -log[H⁺]. <7 acidic, >7 basic.
*3 lines of defense*:
1. *Blood buffers*: HCO₃⁻/CO₂, proteins, phosphate.
2. *Respiratory*: CO₂ excretion via lungs.
3. *Renal*: H⁺ excretion, HCO₃⁻ reabsorption.
*Disorders*:
– *Metabolic acidosis*: Low HCO₃⁻. Causes: lactic acid, ketoacids, renal failure.
– *Respiratory acidosis*: High CO₂. Causes: hypoventilation.
– *Metabolic alkalosis*: High HCO₃⁻. Causes: vomiting, diuretics.
– *Respiratory alkalosis*: Low CO₂. Causes: hyperventilation.
*pH effects on cells*:
– *Enzymes/proteins*: Each has optimal pH. Wrong pH → charge change → denaturation.
– *Membranes*: Only neutral molecules cross easily. Ion trap in lysosomes.
– *ATP synthesis*: Proton gradient drives ATP synthase.
– *Signaling*: Small pH shifts trigger growth, division, apoptosis.
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*Key takeaways for exam*:
1. Know the difference between differential vs isopycnic centrifugation.
2. Memorize water distribution in a 70kg man.
3. Be able to classify acidosis/alkalosis as metabolic vs respiratory.
4. Explain why pH matters for enzyme activity and membrane transport.
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