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Thursday, April 30, 2009

Pharmacokinetics

Pharmacokinetics is the study of what the body does to a drug.
Pharmacodynamics is the study of what a drug does to the body.
Routes of Drug Administration:
Intravenous
Oral
Buccal
Sublingual
Rectal
Intramuscular
Transdermal
Subcutaneous
Inhalational
Topical
Of all of these routes you are most likely to be asked about the transdermal, as it is fashionable.
Otherwise, most other basic pharmacology questions tend to concern the pharmacology of intravenous agents; that is what is discussed below.
First Order Kinetics:
A constant fraction of the drug in the body is eliminated per unit time. The rate of elimination is proportional to the amount of drug in the body. The majority of drugs are eliminated in this way.

What follows concerns drugs which follow first order kinetics.

The Volume of Distribution (Vd) is the amount of drug in the body divided by the concentration in the blood. Drugs that are highly lipid soluble, such as digoxin, have a very high volume of distribution (500 litres). Drugs which are lipid insoluble, such as neuromuscular blockers, remain in the blood, and have a low Vd.
The Clearance (Cl) of a drug is the volume of plasma from which the drug is completely removed per unit time. The amount eliminated is proportional to the concentration of the drug in the blood.
The fraction of the drug in the body eliminated per unit time is determined by the elimination constant (kel). This is represented by the slope of the line of the log plasma concentration versus time.
Cl = kel x Vd
Rate of elimination = clearance x concentration in the blood.
Elimination half life (t1/2): the time taken for plasma concentration to reduce by 50%. After 4 half lives, elimination is 94% complete.
It can be shown that the kel = the log of 2 divided by the t1/2 = 0.693/t1/2.
Likewise, Cl = kel x Vd, so, Cl = 0.693Vd/t1/2.
And t1/2 = 0.693 x Vd / cl
The rate of elimination is the clearance times the concentration in the plasma
Roe = Cl x Cp
Fraction of the total drug removed per unit time = Cl/Vd.
If the volume of distribution is increased, then the kel will decrease, the t1/2 will increase, but the clearance won't change.
Confused?
Example: You have a 10ml container of orange squash. You put this into a litre (ok 990ml!) of water. The Vd of the orange squash is 1000ml. If, each minute, you empty 10ml of the orange liquid into the 10ml container, discard this, and replace it with 10ml of water. The clearance is 10 ml per minute. The elimination half life is: 70 minutes . The kel is Cl/Vd = 10/1000 = 0.01. Shown the other way, 0.693/50 = 0.01.
If the volume of the container is increased to 2000ml, then the clearance remains the same, but the Vd, and consequently the t1/2, increases (to 140 minutes).
Simple, isn't it?
What is described above is a single compartment model, what would occur if the bloodstream was the only compartment in the body (or if the Vd = the blood volume). But the human body is more complex than this: there are many compartments: muscle, fat, brain tissue etc. In order to describe this, we use multicompartment models.
Multicompartment Models:
Why does a patient wake up after 5 minutes after an injection of thiopentone when we know that it takes several hours to eliminate this drug from the body? What happens is that, initially the drug is all in the blood and this blood goes to "vessel rich" organs; principally the brain. After a few minutes the drug starts to venture off into other tissues (fat, muscle etc) it redistributes, the concentration in the brain decreases and the patient wakes up! The drug thus redistributes into other compartments.
If you were to represent this phenomenon graphically, you would follow a picture of rapid fall in blood concentration, a plateau, and then a slower gradual fall. The first part is the rapid redistribution phase, the alpha phase, the plateau is the equilibrium phase (where blood concentration = tissue concentration), and the slower phase, the beta phase, is the elimination phase where blood and tissue concentrations fall in tandem. This is a simple two compartment model and is as much as you need to know.

An couple of interesting pieces of information can be derived from the log concentration versus time graph. If you extrapolate back the elimination line to the y axis, then you get to a point called the CP0 - a theoretical point representing the concentration that would have existed at the start if the dose had been instantly distributed (dose/Vd). From this new straight line you can figure out how long it takes for the concentration to drop by 50%: the elimination half life. Likewise, a similar procedure can be performed on the α phase: the redistribution half life.
While it is very important that you understand these concepts, the reality is that most drugs are infinitely more complicated that this, and computer calculations are required to derive this data.
Bioavailability
This is the fraction of the administered dose that reaches the systemic circulation. Bioavailability is 100% for intravenous injection. It varies for other routes depending on incomplete absorption, first pass hepatic metabolism etc. Thus one plots plasma concentration against time, and the bioavailability is the area under the curve.
Zero Order Elimination
Why if I have 10 pints of beer before midnight will I fail a breathalyser test at 8 am the following morning? Either this is due to alcohol having a very long half life (which it does not) or that alcohol is cleared in a different way.
What happens is that the metabolic pathways responsible for alcohol metabolism are rapidly saturated and that clearance is determined by how fast these pathways can work. The metabolic pathways work to their limit. This is known as zero order kinetics: a constant amount of drug is eliminated per unit time. This form of kinetics occours with several important drugs at high dosage concentrations: phenytoin, salicylates, theophylline, and thiopentone (at very large doses). Because high dose thio is very slow to clear, we no longer use it in infusion for status epilepticus (as it takes ages for the patient to wake up!).
Dosage regimens
The strategy for treating patients with drugs is to give sufficient amounts that the required theraputic effect arises, but not a toxic dose.
The maintenance dose is equal to the rate of elimination at steady state (i.e.at steady state, rate of elimination = rate of administration):
Dosing rate = clearance x desired plasma concentration.
Drugs will accumulate within the body if the drug has not been fully eliminated before the next dose. Steady state concentration is thus arrived at after four half lives. This is all very well if you are willing to wait 4 half lives for the drug to be fully effective, but what if you are not? What you may need to do is to "load" the volume of distribution with the drug to achieve target plasma concentrations rapidly: the loading dose.
The loading dose = the volume of distribution x the desired concentration (i.e. the concentration at steady state).
You can figure this out by: Loading dose = usual maintenance dose / usual dosage interval x kel (t1/2/0.693).
Hepatic Drug Clearance
Many drugs are extensively metabolised by the liver. The rate of elimination depends on 1) The liver's inherent ability to metabolise the drug, 2) the amount of drug presented to the liver for metabolism. This is important because drugs administered orally are delivered from the gut to the portal vein to the liver: the liver gobbles up a varying chunk of the administered drug (pre-systemic elimination) and less is available to the body for theraputic effect. This is why you have to give a higher dose of morphine, for examole, orally, than intravenously.
Hepatic drug clearance (i.e. the amount of each drug gobbled up by the liver) depends on:
1) The Intrinsic clearance (Cl int).
2) Hepatic blood flow.
These two factors are independent of one another, and their combined effect is the proportion of drug gobbled up: the extraction ratio.
For drugs that have a low intrinsic clearance, this effect can be increased by giving a second agent that boosts the effect of the liver's enzyme system; these are enzyme inducers. Examples of such drugs are cigarrettes, antiepileptics (carbamazepine & phenytoin), rifampicin, griseofulvin, alcohol and spironolactone (CAR GAS) [also barbiturates]. Consequnetly if a drug addict is given rifampicin or tuberculosis, a higher dose of heroin is required for the same effect. Enzyme inhibitors have the opposite effect: examples are flagyl, allopurinol, cimetidine, erythromycin, dextropropoxyphene, imipramine, (the) pill (FACE DIP).
Likewise, if the blood flow increases, the liver has less chance to gobble up the drug, and the extraction ratio falls. This is particularly the case, as you would expect, of the intrinsic clearance is low.
Illustration: Think of factory workers picking bad apples out of a pile on a conveyor belt, if only one person (low intrinsic clearance) is doing the picking and the speed of the conveyor belt is increased, more bad apples get through. If there are several pickers (high intrinsic clearance) then they are much more able to cope with an increase in the speed of the conveyor belt, but there will come a rate at which they will become overwhelmed, and bad apples will get through.
From this example you can take home this message: for drugs with a low extraction ratio, the kinetics (the body's ability to deal with the drug) depends on enzymatic activity (giving an enzyme inducer effectively gives the single picker 4 arms!). For high extraction ratio drugs, kinetics depends on liver blood flow - the slower the flow the higher the extraction, the higher the flow the lower the extraction ratio.
Drug distribution
When a drug is introduced into the body, where it ends up depends on a number of factors:
1) blood flow, tissues with the highest blood flow receive the drug first, 2) protein binding, drugs stuck to plasma proteins are crippled, they can only go where the proteins go (and that's not very far!), 3) lipid solubility and the degree of ionisation, this describes the ability of drugs to enter tissues (highly lipid soluble / unionised drugs can basically go anywhere).
Protein Binding
Most drugs bind to proteins, either albumin or alpha-1 acid glycoprotein (AAG), to a greater or lesser extent. Drugs prefer to be free, it is in this state that they can travel throughout the body, in and out of tissues and have their biological effect. The downside of this is that they are easy prey for metabolising enzymes.
As you would expect, more highly bound drugs have a longer duration of action and a lower volume of distribution. Generally high extraction ratio drugs' clearance is high because of low protein binding and, conversely, low extraction ratio drugs' clearance is strongly dependent on the amount of protein binding.
Why is this important? If a drug is highly protein bound, you need to give loads of it to get a theraputic effect; as so much is stuck to protein. But what happens if another agent comes along and starts to compete with the drug for the binding site on the protein? Yes, you guessed it, the amount of free drug is increased. This is really important for drugs that are highly protein bound: if a drug is 97% bound to albumin and there is a 3% reduction in binding (displaced by another drug), then the free drug concentration doubles; if a drug is 70% bound and there is a 3% reduction in binding, this will make little difference.
The drugs that you really need to keep an eye on are: warfarin, diazepam, propranolol and phenytoin. For example, a patient on warfarin is admitted with seizures, you treat the patient with phenytoin, next thing you know - his INR is 10.
The amount of albumin does not appear to be hugely relavent. In disease states such as sepsis, the serum albumin drops drastically, but the free drug concentration does not appear to increase
Degree of ionisation
This is really important with regard to local anaesthetics. The essential fact to know is that highly ionized drugs cannnot cross lipid membranes (basically they can't go anywhere) and unionised drugs can cross freely. Morphine is highly ionised, fentanyl is the opposite. Consequently the latter has a faster onset of action. The degree of ionisation depends on the pKa of the drug and the pH of the local environment. The pKa is the the pH at which the drug is 50% ionised. Most drugs are either weak acids or weak bases. Acids are most highly ionised at a high pH (i.e. in an alkaline environment). Bases are most highly ionised in an acidic environment (low pH). For a weak acid, the more acidic the environment, the less ionised the drug, and the more easily it crosses lipid membranes. If you take this acid, at pKa it is 50% ionised, if you add 2 pH points to this (more alkaline), it becomes 90% ionised, if you reduce the pH (more acidic) by two units, it becomes 10% ionised. Weak bases have the opposite effect.
Local anaesthetics are weak bases: the closer the pKa of the local anaesthetic to the local tissue pH, the more unionised the drug is. That is why lignocaine(pKa 7.7) has a faster onset of action than bupivicaine (pKa 8.3). If the local tissues are alkalinised (e.g. by adding bicarbonate to the local anaesthetic), then the tisssue pH is brought closer to the pKa, and the onset of action is hastened.

Xenobiotic metabolism

Xenobiotic metabolism is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism's normal biochemistry, such as drugs and poisons. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds; however, in some cases, the intermediates in xenobiotic metabolism can themselves be the cause of toxic effects.
Xenobiotic metabolism is divided into three phases. In phase I, enzymes such as cytochrome P450 oxidases introduce reactive or polar groups into xenobiotics. These modified compounds are then conjugated to polar compounds in phase II reactions. These reactions are catalysed by transferase enzymes such as glutathione S-transferases. Finally, in phase III, the conjugated xenobiotics may be further processed, before being recognised by efflux transporters and pumped out of cells.
The reactions in these pathways are of particular interest in medicine as part of drug metabolism and as a factor contributing to multidrug resistance in infectious diseases and cancer chemotherapy. The actions of some drugs as substrates or inhibitors of enzymes involved in xenobiotic metabolism are a common reason for hazardous drug interactions. These pathways are also important in environmental science, with the xenobiotic metabolism of microorganisms determining whether a pollutant will be broken down during bioremediation, or persist in the environment.

MTT assay

The MTT assay and the MTS assay are laboratory tests and standard colorimetric assays (an assay which measures changes in color) for measuring the activity of enzymes that reduce MTT or MTS + PMS to formazan, giving a purple color. It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials, since those agents would result in cell toxicity and therefore metabolic dysfunction and therefore decreased performance in the assay.
Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in living cells.[1] A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The absorption maximum is dependent on the solvent employed.
MTS is a more recent alternative to MTT. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline.[2] It is advantageous over MTT in that (1) the reagents MTS + PMS are reduced more efficiently than MTT, and (2) the product is water soluble, decreasing toxicity to cells seen with an insoluble product.
These reductions take place only when reductase enzymes are active, and therefore conversion is often used as a measure of viable (living) cells. However, it is important to keep in mind that other viability tests (such as the CASY cell counting technology) sometimes give completely different results, as many different conditions can increase or decrease metabolic activity. Changes in metabolic activity can give large changes in MTT or MTS results while the number of viable cells is constant. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve

DNase footprinting assay

A DNase footprinting assay[1] is a DNA footprinting technique from molecular biology/biochemistry that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme, deoxyribonuclease (DNase, for short) to cut the radioactively end-labelled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern.
For example, the DNA fragment of interest may be PCR amplified using a 32P 5' labeled primer, with the end result being many DNA molecules with a radioactive label on one end of one strand of each double stranded molecule. Cleavage by DNase will produce fragments, the smaller of which will move further on the electrophoretic gel. The fragments which are smaller with respect to the 32P-labelled end will appear further on the gel than the longer fragments. The gel is then used to expose a special photographic film.
The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as the "footprint".
By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed.
This technique was developed by David Galas and Albert Schmitz at Geneva in 1977

Reversible inhibitors

[edit] Enzyme activity
Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical and commonly-used value is 1 enzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds to 16.67 nanokatals.[1]

[edit] Specific activity
The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min-1mg-1). Specific activity gives a measurement of the purity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of enzyme. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-1. Specific activity is a measure of enzyme processivity, usually constant for a pure enzyme.

[edit] Related terminology
The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L − 1s − 1)
The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated.

[edit] Types of assay
All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:[2]
(1) Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.
(2) Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.
(3) Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require rapid mixing and observation techniques.
(4) Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.
Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

Temperature-controlled cuvette holder in a spectrophotometer.

[edit] Continuous assays
Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.

[edit] Spectrophotometric
In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.
UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidised forms. An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.[3]
Direct versus coupled assays

Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase.
Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here, the product of one reaction is used as the substrate of another, easily-detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase.

[edit] Fluorometric
Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light.
An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase.[4] Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase.

[edit] Calorimetric

Chemiluminescence of Luminol
Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assay in any other way.[5]

[edit] Chemiluminescent
Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected.
The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.

[edit] Light Scattering
Static Light Scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate. Hence the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.

[edit] Discontinuous assays
Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.

[edit] Radiometric
Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes most frequently used in these assays are 14C, 32P, 35S and 125I. Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).
Radioactivity is usually measured in these procedures using a scintillation counter.

[edit] Chromatographic
Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher than HPLC instruments (see HPLC#Pump_pressure).[6]

[edit] Factors to control in assays
Salt Concentration: Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria.
Effects of Temperature: All enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme. The "optimum" temperature for human enzymes is usually between 35 and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C. Enzymes from thermophilic archaea found in the hot springs are stable up to 100 °C.[7] However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it would give you low activity at these temperatures.
Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.
Substrate Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau.
Level of crowding, large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding.[8]

[edit] List of enzyme assays
MTT assay
Overlay assay
Fluorescein diacetate hydrolysis

[edit] See also
Restriction enzyme
DNase footprinting assay
Enzyme kinetics

[edit] References
^ Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1979). "Units of Enzyme Activity". Eur. J. Biochem. 97: 319–20. doi:10.1111/j.1432-1033.1979.tb13116.x. http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1432-1033.1979.tb13116.x.
^ Schnell, S., Chappell, M. J., Evans, N. D. and Roussel, M. R. The mechanism distinguishability problem in biochemical kinetics: The single-enzyme, single-substrate reaction as a case study. Comptes Rendus Biologies 2006; 329, 51-61. DOI: 10.1016/j.crvi.2005.09.005
^ Bergmeyer, H. U. "Methods of Enzymatic Analysis", Vol. 4, Academic Press (New York, NY:1974), pp.2066-2072.
^ Passonneau, J. V., and Lowry, O. H. "Enzymatic Analysis. A Practical Guide", Humana Press (Totowa, NJ:1993), pp.85-110.
^ Todd MJ, Gomez J. Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem. 2001 Sep 15;296(2):179-87.
^ Churchwella, M; Twaddlea, N; Meekerb, L; and Doergea, D. Improving Sensitivity In Liquid Chromatography-Mass Spectrometry. Journal of Chromatography B Volume 825, Issue 2, 25 October 2005, Pages 134-143
^ Cowan DA (1997). "Thermophilic proteins: stability and function in aqueous and organic solvents". Comp. Biochem. Physiol. A Physiol. 118 (3): 429–38. doi:10.1016/S0300-9629(97)00004-2. PMID 9406427.
^ Minton AP (2001). "The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media". J. Biol. Chem. 276 (14): 10577–80. doi:10.1074/jbc.R100005200. PMID 11279227. http://www.jbc.org/cgi/content/full/276/14/10577.
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Principles of pathology
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Cell death: Necrosis (Liquefactive necrosis, Coagulative necrosis, Caseous necrosis) - Apoptosis - Pyknosis - Karyorrhexis - Karyolysis
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vdeProteins: key methods of study
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Protein purification - Green fluorescent protein - Western blot - Protein immunostaining - Protein sequencing - Gel electrophoresis/Protein electrophoresis - Protein immunoprecipitation - Peptide mass fingerprinting
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Protein structure prediction - Protein-protein docking - Protein structural alignment - Protein ontology - Protein-protein interaction prediction
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Enzyme assay - Protein assay - Secretion assay
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Sunday, April 26, 2009

Enzyme inhibitor
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HIV protease in a complex with the protease inhibitor ritonavir. The structure of the protease is shown by the red, blue and yellow ribbons. The inhibitor is shown as the smaller ball-and-stick structure near the centre. Created from PDB 1HXW.
Enzyme inhibitors are molecules that bind to enzymes and decrease their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity.
The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalysing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Many drug molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology. A medicinal enzyme inhibitor is often judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed to inhibit the enzyme). A high specificity and potency ensure that a drug will have few side effects and thus low toxicity.
Enzyme inhibitors also occur naturally and are involved in the regulation of metabolism. For example, enzymes in a metabolic pathway can be inhibited by downstream products. This type of negative feedback slows flux through a pathway when the products begin to build up and is an important way to maintain homeostasis in a cell. Other cellular enzyme inhibitors are proteins that specifically bind to and inhibit an enzyme target. This can help control enzymes that may be damaging to a cell, such as proteases or nucleases; a well-characterised example is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein–protein interactions.[1] Natural enzyme inhibitors can also be poisons and are used as defenses against predators or as ways of killing prey.

Saturday, April 18, 2009

[edit] Use of carbohydrates as an energy source
See also carbohydrate metabolism
Glucose is the major energy source in most life forms. For instance, polysaccharides are broken down into their monomers (glycogen phosphorylase removes glucose residues from glycogen). Disaccharides like lactose or sucrose are cleaved into their two component monosaccharides.

[edit] Glycolysis (anaerobic)
Glucose is mainly metabolized by a very important and ancient ten-step pathway called glycolysis, the net result of which is to break down one molecule of glucose into two molecules of pyruvate; this also produces a net two molecules of ATP, the energy currency of cells, along with two reducing equivalents in the form of converting NAD+ to NADH. This does not require oxygen; if no oxygen is available (or the cell cannot use oxygen), the NAD is restored by converting the pyruvate to lactate (lactic acid) (e. g. in humans) or to ethanol plus carbon dioxide (e. g. in yeast). Other monosaccharides like galactose and fructose can be converted into intermediates of the glycolytic pathway.

[edit] Aerobic
In aerobic cells with sufficient oxygen, like most human cells, the pyruvate is further metabolized. It is irreversibly converted to acetyl-CoA, giving off one carbon atom as the waste product carbon dioxide, generating another reducing equivalent as NADH. The two molecules acetyl-CoA (from one molecule of glucose) then enter the citric acid cycle, producing two more molecules of ATP, six more NADH molecules and two reduced (ubi)quinones (via FADH2 as enzyme-bound cofactor), and releasing the remaining carbon atoms as carbon dioxide. The produced NADH and quinol molecules then feed into the enzyme complexes of the respiratory chain, an electron transport system transferring the electrons ultimately to oxygen and conserving the released energy in the form of a proton gradient over a membrane (inner mitochondrial membrane in eukaryotes). Thereby, oxygen is reduced to water and the original electron acceptors NAD+ and quinone are regenerated. This is why humans breathe in oxygen and breathe out carbon dioxide. The energy released from transferring the electrons from high-energy states in NADH and quinol is conserved first as proton gradient and converted to ATP via ATP synthase. This generates an additional 28 molecules of ATP (24 from the 8 NADH + 4 from the 2 quinols), totaling to 32 molecules of ATP conserved per degraded glucose (two from glycolysis + two from the citrate cycle). It is clear that using oxygen to completely oxidize glucose provides an organism with far more energy than any oxygen-independent metabolic feature, and this is thought to be the reason why complex life appeared only after Earth's atmosphere accumulated large amounts of oxygen.

[edit] Gluconeogenesis
Main article: Gluconeogenesis
In vertebrates, vigorously contracting skeletal muscles (during weightlifting or sprinting, for example) do not receive enough oxygen to meet the energy demand, and so they shift to anaerobic metabolism, converting glucose to lactate. The liver regenerates the glucose, using a process called gluconeogenesis. This process is not quite the opposite of glycolysis, and actually requires three times the amount of energy gained from glycolysis (six molecules of ATP are used, compared to the two gained in glycolysis). Analogous to the above reactions, the glucose produced can then undergo glycolysis in tissues that need energy, be stored as glycogen (or starch in plants), or be converted to other monosaccharides or joined into di- or oligosaccharides. The combined pathways of glycolysis during exercise, lactate's crossing via the bloodstream to the liver, subsequent gluconeogenesis and release of glucose into the bloodstream is called the Cori cycle
[edit] Carbohydrates
Main articles: Carbohydrates, Monosaccharides, Disaccharides, and Polysaccharides

A molecule of sucrose (glucose + fructose), a disaccharide.
Carbohydrates have monomers called monosaccharides. Some of these monosaccharides include glucose (C6H12O6), fructose (C6H12O6), and deoxyribose (C5H10O4). When two monosaccharides undergo dehydration synthesis, water is produced, as two hydrogen atoms and one oxygen atom are lost from the two monosaccharides' carboxyl group.

[edit] Lipids
Main articles: Lipids, Glycerol, and Fatty acids

A triglyceride with a glycerol molecule on the left and three fatty acids coming off it.
Lipids are usually made up of a molecule of glycerol and other molecules. In triglycerides, or the main lipid, there is one molecule of glycerol, and three fatty acids. Fatty acids are considered the monomer in that case, and could be saturated or unsaturated. Lipids, especially phospholipids, are also used in different pharmaceutical products, either as co-solubilisers e.g. in Parenteral infusions or else as drug carrier components (e.g. in a Liposome or Transfersome).

[edit] Proteins
Main articles: Proteins and Amino Acids

The general structure of an α-amino acid, with the amino group on the left and the carboxyl group on the right.
Proteins are large molecules, and have monomers of amino acids. There are 20 different known kinds of amino acids, and they contain a carboxyl group, an amino group, and an "R" group. The "R" group is what makes each amino acid different. When Amino acids combine, they form a special bond called a peptide bond, and become a polypeptide, or a protein.

[edit] Nucleic Acids
Main articles: Nucleic acid, DNA, RNA, and Nucleotides

The structure of deoxyribonucleic acid (DNA), the picture shows the monomers being put together.
Nucleic acids are very important in biochemistry, as they are what make up DNA, something all cellular organism use to store their genetic information. The most common nucleic acids are deoxyribonucleic acid and ribonucleic acid. Their monomers are called nucleotides. The most common nucleotides are called adenine, cytosine, guanine, thymine, and uracil. Adenine binds with thymine and uracil, thymine only binds with adenine, and cytosine and guanine can only bind with each other.

[edit] Carbohydrates
Main article: Carbohydrate
The function of carbohydrates includes energy storage and providing structure. Sugars are carbohydrates, but not all carbohydrates are sugars. There are more carbohydrates on Earth than any other known type of biomolecule; they are used to store energy and genetic information, as well as play important roles in cell to cell interactions and communications.

[edit] Monosaccharides

Glucose
The simplest type of carbohydrate is a monosaccharide, which among other properties contains carbon, hydrogen, and oxygen, mostly in a ratio of 1:2:1 (generalized formula CnH2nOn, where n is at least 3). Glucose, one of the most important carbohydrates, is an example of a monosaccharide. So is fructose, the sugar that gives fruits their sweet taste. Some carbohydrates (especially after condensation to oligo- and polysaccharides) contain less carbon relative to H and O, which still are present in 2:1 (H:O) ratio. Monosaccharides can be grouped into aldoses (having an aldehyde group at the end of the chain, e. g. glucose) and ketoses (having a keto group in their chain; e. g. fructose). Both aldoses and ketoses occur in an equilibrium between the open-chain forms and (starting with chain lengths of C4) cyclic forms. These are generated by bond formation between one of the hydroxyl groups of the sugar chain with the carbon of the aldehyde or keto group to form a hemiacetal bond. This leads to saturated five-membered (in furanoses) or six-membered (in pyranoses) heterocyclic rings containing one O as heteroatom.

[edit] Disaccharides

Sucrose: ordinary table sugar and probably the most familiar carbohydrate.
Two monosaccharides can be joined together using dehydration synthesis, in which a hydrogen atom is removed from the end of one molecule and a hydroxyl group (—OH) is removed from the other; the remaining residues are then attached at the sites from which the atoms were removed. The H—OH or H2O is then released as a molecule of water, hence the term dehydration. The new molecule, consisting of two monosaccharides, is called a disaccharide and is conjoined together by a glycosidic or ether bond. The reverse reaction can also occur, using a molecule of water to split up a disaccharide and break the glycosidic bond; this is termed hydrolysis. The most well-known disaccharide is sucrose, ordinary sugar (in scientific contexts, called table sugar or cane sugar to differentiate it from other sugars). Sucrose consists of a glucose molecule and a fructose molecule joined together. Another important disaccharide is lactose, consisting of a glucose molecule and a galactose molecule. As most humans age, the production of lactase, the enzyme that hydrolyzes lactose back into glucose and galactose, typically decreases. This results in lactase deficiency, also called lactose intolerance.
Sugar polymers are characterised by having reducing or non-reducing ends. A reducing end of a carbohydrate is a carbon atom which can be in equilibrium with the open-chain aldehyde or keto form. If the joining of monomers takes place at such a carbon atom, the free hydroxy group of the pyranose or furanose form is exchanged with an OH-side chain of another sugar, yielding a full acetal. This prevents opening of the chain to the aldehyde or keto form and renders the modified residue non-reducing. Lactose contains a reducing end at its glucose moiety, whereas the galactose moiety form a full acetal with the C4-OH group of glucose. Saccharose does not have a reducing end because of full acetal formation between the aldehyde carbon of glucose (C1) and the keto carbon of fructose (C2).

[edit] Oligosaccharides and polysaccharides

Cellulose as polymer of β-D-glucose
When a few (around three to six) monosaccharides are joined together, it is called an oligosaccharide (oligo- meaning "few"). These molecules tend to be used as markers and signals, as well as having some other uses. Many monosaccharides joined together make a polysaccharide. They can be joined together in one long linear chain, or they may be branched. Two of the most common polysaccharides are cellulose and glycogen, both consisting of repeating glucose monomers.
Cellulose is made by plants and is an important structural component of their cell walls. Humans can neither manufacture nor digest it.
Glycogen, on the other hand, is an animal carbohydrate; humans and other animals use it as a form of energy storage.
[edit] History
Main article: History of biochemistry
Originally, it was generally believed that life was not subject to the laws of science the way non-life was. It was thought that only living beings could produce the molecules of life (from other, previously existing biomolecules). Then, in 1828, Friedrich Wöhler published a paper on the synthesis of urea, proving that organic compounds can be created artificially.[2][3]
The dawn of biochemistry may have been the discovery of the first enzyme, diastase (today called amylase), in 1833 by Anselme Payen. Eduard Buchner contributed the first demonstration of a complex biochemical process outside of a cell in 1896: alcoholic fermentation in cell extracts of yeast. Although the term “biochemistry” seems to have been first used in 1882, it is generally accepted that the formal coinage of biochemistry occurred in 1903 by Carl Neuberg, a German chemist. Previously, this area would have been referred to as physiological chemistry. Since then, biochemistry has advanced, especially since the mid-20th century, with the development of new techniques such as chromatography, X-ray diffraction, NMR spectroscopy, radioisotopic labeling, electron microscopy and molecular dynamics simulations. These techniques allowed for the discovery and detailed analysis of many molecules and metabolic pathways of the cell, such as glycolysis and the Krebs cycle (citric acid cycle).
Another significant historic event in biochemistry is the discovery of the gene and its role in the transfer of information in the cell. This part of biochemistry is often called molecular biology. In the 1950s, James D. Watson, Francis Crick, Rosalind Franklin, and Maurice Wilkins were instrumental in solving DNA structure and suggesting its relationship with genetic transfer of information. In 1958, George Beadle and Edward Tatum received the Nobel Prize for work in fungi showing that one gene produces one enzyme. In 1988, Colin Pitchfork was the first person convicted of murder with DNA evidence, which led to growth of forensic science. More recently, Andrew Z. Fire and Craig C. Mello received the 2006 Nobel Prize for discovering the role of RNA interference (RNAi), in the silencing of gene expression
Today, there are three main types of biochemistry as established by Michael E. Sugar. Plant biochemistry involves the study of the biochemistry of autotrophic organisms such as photosynthesis and other plant specific biochemical processes. General biochemistry encompasses both plant and animal biochemistry. Human/medical/medicinal biochemistry focuses on the biochemistry of humans and medical illnesses.

[edit] Monomers and Polymers
Main articles: Monomer and Polymer
Monomers and polymers are a structural basis in which the four main macromolecules (Carbohydrates, lipids, proteins, and nucleic acids), or biopolymers, of biochemistry are based on. Monomers are smaller micromolecules that are put together to make macromolecules. Polymers are those macromolecules that are created when monomers are synthesized together. When they are synthesized, the two molecules undergo a process called dehydration synthesis

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