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Effect of Enzyme Concentration on Protein Digestion

Enzymes were discovered by a German chemist Eduard Buchner near the end of the 19th century. He had been trying to extract a fluid for medicinal use from yeast, however, the yeast extract kept going bad. He then decided to add sugar to the yeast, however, the yeast converted the sugar into alcohol, which is also known as fermentation. Buchner investigated into this and soon found out that living cells were not responsible for this fermentation and that it was caused by the fluid that was trying to be extracted from the yeast. The word enzyme was coined for the active ingredients in the juice that promoted fermentation. Although enzyme literally means “in yeast”, it is now however being used as the collective noun for several hundreds of compounds that have shown to have a catalytic action on specific chemical reactions.
Enzymes are biological or organic catalysts made up of protein. They catalyse (increase/decrease the rate of) chemical reactions without themselves being chemically changed at the end of the reaction. It can therefore be used repeatedly and so is effective in small amounts. They essentially work by lowering the activation energy of the reactions and hence allowing the reaction to place at a quicker rate. In enzymatic reactions, the molecules are the start of the process are called substrates, and the converted molecules, the products.
Properties of enzymes:
Enzymes have the following properties:
Enzymes alter the rate of chemical reactions without themselves being chemically changed at the end of the reaction.
Enzymes are very potent. Since enzymes are very specific, a small amount of an enzyme is capable of catalysing a huge chemical reaction.
Enzymes are affected by temperature. Enzymes are inactive at low temperatures. Increasing the temperature increases the activity of the enzymes. There is an optimum working temperature at which certain enzymes work best. This is normally between 37-42 degree centigrades. However, a high temperature, anything above 45 degree centigrades normally destroys the active sites of the enzymes and causes it to denature. This permanently damages the enzyme and they become functionless.
Enzymes are affected by pH. Certain enzymes work best in acidic conditions whereas certain enzymes function better in alkaline conditions. For example, pepsin works best in the stomach where the pH is below 7, however intestinal enzymes work better in coditions of pH of above 7.
Some enzymes may require a compound to be bound to them before they can catalyse chemical reactions. These compounds are called co-enzymes.
Enzymes can work in either directions. Metabolic reactions are reversible and the direction in which the reaction goes depends on the amounts of substrate and products present. The reaction will proceed from left to right until an equilibrium is reached between the substrates and products. Also, if there is a large amount of products, then the reverse reaction starts and hence causes the product to be split up until again equilibrium is established.
Lock and Key

Negative Staining In Transmission Electron Microscopy

Biologic structures, because of low mass density, show little contrast hence contrast enhancement of such samples is necessary in order to study them. Negative staining is one such contrast enhancement method. Negative staining for light microscopy is an indirect staining where the background instead of the specimen is stained the unstained specimen appears as bright object against a dark background hence the name ‘Negative Staining’ [1]. Similarly in negative staining for transmission electron microscopy {TEM} the specimen is unstained whereas the background appears dark, as the heavy metals used for negative staining do not penetrate the specimen but stain the background dark. Hence here the specimen is electron transparent (unstained) but is surrounded by an electron dense stain unlike positive staining method where the heavy metal ions react with the specimen resulting in an increase in the density and contrast [2].
Negative staining is applicable to viruses, bacteria sub cellular organelles, liposomes, artificial membranes, synthetic DNA array and also to polymer solutions, components of molecular mass in the range of 200 KDa upto several MDa [such as the molluscan, hemocyanin and ribosomes] [3]
After staining the specimen and air drying it, a firm structurless substrate is used to support the specimen. Collodion or formvar substrates stabilized with carbon are generally used [4]. To minimize specimen damage during TEM, a double condenser lens and an anti-contaminator in the specimen area are needed. Often the specimens are not fixed but simply air-dried yet the resolutions obtained are far better than sectioned since thick plastic embedments are absent. As negative stains cover the specimen in a firm, amorphous supporting matrix without reacting with the specimen like chemical fixatives they improve resolution. [4] Negative staining can give a resolution limited to 20-25A however recent studies have revealed that it is possible to preserve and record periodic structural information in catalase crystals to a level of 4.0 A by negative staining [16].
Negative stains A good negative stain reveals the outline and structure as the stain surrounds and fills in depressions in structures providing a hint of three dimensionality. Highest resolution is obtained when the stain is without any graininess. [4] Negative stains are salts heavy metals such as uranium, tungsten which are electron dense stains. Some of the negative stains used are ammonium molybdate, phosphotungstic acid [K-PTA], uranyl magnesium acetate, uranyl oxylate [5]. These stains are generally prepared as 1% or 2% w/v. A low concentration [eg:0.1 mM to 1.0mM ] of neutral n-octyl- -D-glucopyranoside(OQ) can be added to any of the above negative-stain solution to improve spreading and assist in permeation within the specimens.
Uranyl acetate is prepared to give a 1 or 2% (w/v) solution [4] its light sensitivity and yellow in colour. It is used for staining viruses, bacteria, cell fractions, frozen section, macromolecules. Moreover it may have a fixative effect as a cross linker for phospholipids when used at a concentration of 0.5% in 50% ethanol after 0s04 treatment. [6]. It reacts quickly as well stabilizes lipids. Other heavy metal stain commonly Use is phosphotongistic acid or its corresponding Na or K salts. It is used to study viruses, membrane proteins especially suitable to visualize bacterial inclusion bodies and bacterial cells [6]. 1-2% aqueous solution is prepared using 1N KOH. Phosphotungstic acid does not act as a fixative hence treatment with a fixative like aldehyde is a must, and unlike uranyl acetate the sample penetration is slower. An improved staining method was developed as results with uranyl acetate gave unpredictable results as well as can contamination on a stained grid. The method involve dissolving uranyl acetate in methanol to accomplish en bloc staining which stains in a shorter time and gives as better contrast [7].
1 to 2% Ammonium molybdate is another commonly used negative stain for observing enzyme subunits, membranes or cell fractions. Due to its finer background, ammonium molybdate gives a better resolution of the details.
Negative staining procedures The Single Droplet Negative Staining Technique
In this technique a drop of the sample is placed on a formvar / carbon-coated grid via a locking forcep, after waiting for 30 seconds a drop of negative stain is placed on to the grid and the excessed is removed. After drying the sample is ready to be examined.
Flotation Method
A coated grid is floated on a droplet of sample a parafilm or dental wax for 1 minute to permit adsorption of the specimen. Then the grid is transferred to the drop of negative stain for 30 seconds. An alternative to this would be mixing the stain and sample and then floatation of the grid on the mixture.
Imaging by Negative staining. Negative staining due to their higher image contrast remains an important tool for the study of biological macromolecules. Negative staining has been used to study structure of various enzymes like beef liver glutamate dehydrogenase [8], functional and structural analysis of acetylcholine receptor rich membranes [9]. It has also been useful to study morphology of plant mitochondria [10]. Negative staining has also been used in staining of phospholipids for their structural modification by surface active agents. Negative staining has also been used to study HDL3 [11]
Negative staining in virology Negative staining is an important techniques in virology used for studying the morphology of viruses as well as a detection of viruses in medical samples as a rapid diagnosis procedure. The first electron micrograph of poxvirus was published in 1938. Negative staining provides morphological information about symmetry and capsomer arrangement that makes identification of viruses and classification of viruses into morphologically similar groups possible. Human immunodeficiency virus 1 (HIV-1) and HIV-2 by negative staining electron microscopy. HIV-2 cultures contained large numbers of 130-200 nm particles containing a 130-nm-long by 30-70 nm-wide cores. This core is probably of conical or pear-shaped morphology [12] Negative staining electron microscopy was used to study the simian immunodeficiency virus (SlVmac251) which revealed the presence of central core and internal structures [13]. Negative staining was first used in clinical virology for differential diagnosis of smallpox, chicken pox using fluid from the vesicles on the patients’ skin. Negative staining was an important technique during the eradication of the small pox. Negative-contrast electron microscopy is well established as a rapid means of detecting both poxvirus and herpes virus particles in diagnostic specimen materials. The technique is rapid and sensitive, and can be used to detect inactivated virus in diagnostic specimens. However, the technique has several drawbacks, the major one being a lack of specificity as differentiation is possible between the virus groups but not between the viruses within a group [14]. And because electron microscopy is not suitable for screening large numbers of samples, many alternate immunologic molecular and biochemical methods for rapid detection of large number of samples In conclusion, though this technique was once used on a large scale for diagnostic virology it remains useful for the occasional identification of unknown agents during particular outbreaks.

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