BIO.docx

BIO Biochemistry All substances are made from **atoms** of **elements**. **Molecules** are made from two or more atoms. - If the *atoms* in the molecule are *the same*, then the molecule is that of an **element**. - If the *atoms* are *different*, then the molecule is a **compound**. *...

BIO Biochemistry All substances are made from **atoms** of **elements**. **Molecules** are made from two or more atoms. - If the *atoms* in the molecule are *the same*, then the molecule is that of an **element**. - If the *atoms* are *different*, then the molecule is a **compound**. **Ions** can be formed from metals, non-metals, or combinations of elements. Ions can be: - **positively charged** -- they have **lost** one or more electrons and have more protons than electrons, which is why they have a positive charge - **negatively charged** -- they have **gained** one or more electrons and have more electrons than protons, which is why they have a negative charge. **Compounds** are made from atoms of **two or more elements** and can include metals and non-metals (**ionic compounds**) or just non-metals (**molecular compounds**). While ions are all charged, molecules can have: - **no charge** -- **non-polar** - or a **slight charge** -- **polar**. Particles with a charge (ions or polar molecules) have different properties from molecules with no charge (non-polar). - Ions and polar compounds **attract** oppositely charged particles and play important roles in the structure of molecules. - Non-polar compounds do not dissolve in water but will dissolve in lipids (fats/oils) -- they are said to be **lipid-soluble**. **Elements in living organisms** The six most common elements, accounting for 99% of the mass of the human body are shown in the table below. ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Element** **Percentage of human body mass** **Function** --------------------------------- ----------------------------------- ------------------------------------------------------------------------------------------------------------------- **Oxygen**\ 65%\ Carbon, hydrogen and oxygen are the main components of all organic molecules. Found in amino acids/nucleic acids. **Carbon**\ 18%\ **Hydrogen**\ 10%\ **Nitrogen** 3% **Calcium** **2%** **Strengthens teeth, bones and nerves in animals, and cell walls in plants**. **Phosphorus** (*as phosphate*) **1%** **Present in cell membranes/ATP/nucleic acids.** ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- The remaining 1% of the mass of the human body is made of about 10 other elements, mainly: **Element** **Percentage of human body mass** **Function** --------------- ----------------------------------- ---------------------------------------------------------- Potassium 0.35% Nerve impulse transmission Sulfur 0.25% Some amino acids Chlorine 0.15% Carbon dioxide transportn Sodium 0.15% Nerve impulse transmission **Magnesium** **0.05%** **Enzyme function (and chlorophyll function in plants)** **Iron** **0.004%** **Oxygen transport** Copper trace Enzyme function Manganese trace Enzyme function Zinc trace Enzyme function Iodine trace Growth hormone function **Ions** Most of these key elements are found as **inorganic ions**. **Positive ions** **Negative ions** ----------------------- ---------------------------------- Mg*X*2+ Phosphate, PO*X*4 *X*3− Fe*X*2+ Sulfate, SO*X*4 *X*2− K*X*+ Nitrate, NO*X*3 *X*− Na*X*+ Chloride, Cl*X*− Ca*X*2+ Hydrogen carbonate, HCO*X*3 *X*− Ammonium, NH*X*4 *X*+ **Organic compounds** always contain the elements **carbon** and **hydrogen**, and many contain **oxygen** and/or **nitrogen**. For example: - glucose, C*X*6 H*X*12 O*X*6 - glycine (an amino acid), C*X*2 H*X*5 NO*X*2. Organic compounds are usually produced by living organisms or from the decay of dead organisms (remember crude oil and coal formation from GCSE). **Inorganic compounds** can also contain **carbon**, **hydrogen**, **oxygen** and **nitrogen**, but can be made without the involvement of living organisms. For example: - carbon dioxide, CO*X*2 - water, H*X*2 O - hydrogen carbonate ions, HCO*X*3 *X*−. This topic will focus on **biochemicals** -- these are the compounds made by and found in living organisms: - carbohydrates - lipids - proteins. Water is the most abundant compound in any organism (about 60-70% of the fresh mass of a human). Water is essential as all **biochemical reactions** take place in **aqueous solution** i.e., dissolved in water. Water is a **polar** molecule -- it has no overall charge, but the hydrogen atoms have a partial **positive** charge and the oxygen atoms have a partial **negative** charge. A water molecule is usually drawn using **solid** lines for the bonds between the hydrogen and oxygen atoms. The partial charges are shown as *δ*+, delta positive and *δ*−, delta negative. Because of their **polarity**, water molecules attract each other by forming **hydrogen bonds**. This is usually represented by a series of vertical lines as shown in the diagram. Because of their polarity, water molecules are attracted to other water molecules and charged particles. This helps charged particles dissolve in water. For this reason, water is sometimes referred to as the **universal solvent** as a large number of substances can dissolve in water. Water is also important in chemical reactions, as many small organic molecules can be combined with the loss of a water molecule. **Condensation reactions** and large molecules are often broken down by **hydrolysis** which requires the addition of a water molecule. **Significance of water for life on Earth** In addition to water acting as a solvent and its involvement in biochemical reactions, it also has other properties that make it essential for life on Earth. **Properties water** **Significance for life** ----------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Ice is less dense than water. This forms an insulating layer over the surface of aquatic habitats; ponds and other aquatic habitats do not freeze solid so animals can still move/swim. Water is liquid at most temperatures found on earth. It can be used as a transport medium e.g., in blood in mammals, water transports dissolved ions up the xylem in plants. Water is colourless/transparent. Light can pass through for aquatic plants to photosynthesise; light can pass through the cytoplasm of plant cells so it can reach the chloroplasts. Water has a high surface tension. The surface of water can support the mass of many organisms and becomes a habitat for them e.g., pond skaters. Water has a high specific heat capacity -- it can absorb a lot of energy with only a small rise in temperature. The temperature of cells and aquatic habitats does not change quickly -- conditions thermally remain stable. This is useful inside cells as the temperature of the cytoplasm is stable so enzymes do not denature. Water has a high latent heat of vapourisation. A lot of energy is needed to evaporate water so organisms use water evaporation to cool down (e.g., sweating) and aquatic habitats don't disappear easily by evaporation. Transpiration in plants also has a cooling effect on leaves. Water has strong cohesive and adhesive properties. Due to hydrogen bonds, water molecules stick together (cohesion) and stick to other non-polar or charged substances (adhesion) -- so water molecules can be placed under high tensile (pulling) forces and pulled through plants during transpiration. **Monosaccharides** are **monomers** -- single sugars named according to the number of carbon atoms in the molecule. The numbers on the diagrams below show the number given to each carbon atom in the molecules. All carbohydrates contain the elements **carbon**, **hydrogen** and **oxygen**. Monosaccharides have the **general formula**: (CH*X*2 O)*Xn* or C*Xn* (H*X*2 O)*Xn* or C*Xn* H*X*2*n* O*Xn* where n = the number of atoms e.g., (CH*X*2 O)*X*6 =C*X*6 (H*X*2 O)*X*6 =C*X*6 H*X*12 O*X*6. **Structural isomers** Molecules with the same molecular formula but with different arrangements of their atoms are called **structural isomers**. In the diagram, glucose, galactose and fructose all have the same molecular formula, C*X*6 H*X*12 O*X*6. So, they are **structural isomers**. However, ribose and deoxyribose have different molecular formulae so cannot be structural isomers: - ribose, C*X*5 H*X*10 O*X*5 - deoxyribose, C*X*5 H*X*10 O*X*4. Many monosaccharides can exist in **straight chain forms** or **ring forms**, and can also show **alpha (α)** and **beta (β)** isomerism. The only difference in the **alpha (α)** and **beta (β)** ring isomers is the position of the OH group on carbon atom 1. To remember which ring form is which, use **ABBA**: **A**lpha OH **B**elow -- **B**eta OH **A**bove Different isomers of glucose are shown in the diagram. You need to be able to recognise the following hexose sugars. **Fructose** has a central ring of four carbon atoms and one oxygen atom, with a CH*X*2 OH at carbon atoms 1 and 6. **Glucose** and **galactose** all have a central ring of five carbon atoms and one oxygen atom, with a CH*X*2 OH group at carbon atom 6. They can also exist in alpha and beta forms. Use the table below to tell the difference between the different sugars. **Position of** OH **on carbon atom** **Alpha glucose** **Beta glucose** **Alpha galactose** **Beta galactose** --------------------------------------- ------------------- ------------------ --------------------- -------------------- **1** below above below above **2** below below above above **3** above above below below **4** below below below below Disaccharides are sugars made from two monosaccharide units. They are formed by a condensation reaction (i.e., loss of water) from OH groups on two monosaccharides. The bond that holds them together is called a glycosidic bond. The glycosidic bond can be broken by the chemical insertion of water -- this reforms the OH groups and is called a hydrolysis reaction. The name of the bond depends on: whether the bond lies above or below the carbon atom: α/alpha above β/beta below the numbers of the carbon atoms that the OH groups are attached to. In maltose the bond lies below C1 and water is lost from C1 on one glucose and C4 on the second. So, the name of the bond is an α-1-4 glycosidic bond. The diagram shows the formation of a glycosidic bond to make a molecule of maltose and the breaking of this bond when the molecule is hydrolysed. Lactose and sucrose are two other disaccharides that you need to be able to recognise. These are shown in the diagram below. Note: since fructose has the same group (CHX2 OH) at both ends of the carbon chain, the OH could be on carbon 2 or carbon 5 -- in this case, use the smallest number. **Polysaccharides** are **complex carbohydrates**. They are large molecules, or polymers, consisting of chains of monosaccharides linked together by **glycosidic bonds**. Some polysaccharides have **metabolic** functions and others have **structural** functions in cells and organisms. ***Starch and glycogen*** are both examples of carbohydrates that are involved in the **metabolism** of an organism. They are both **storage** polysaccharides that can store and release glucose as necessary. Both *starch (**plants**)* and *glycogen (**animals***) are made of chains of *α-glucose*. **Starch** is a mixture of two different polysaccharides, **amylose** (which forms **coiled** molecules) and **amylopectin** (a branched molecule). In amylopectin, the bonds between glucose molecules within a branch are α-1,4 glycosidic bonds but at branching points, the bonds are α-1,6 glycosidic bonds. Glucose is a **polar** molecule -- **hydrogen bonds** can form between the O^δ-^ on C2 of one glucose molecule and C3^δ+^ of the next glucose molecule in the chain. As a result, the amylose molecule *coils up* to form a **helix**. This makes starch a compact molecule that is less soluble in water -- ideal properties for storage of glucose. Also, because starch is insoluble it *does not affect the water potential* of the cell in which it is stored. This means that starch is osmotically stable. **Glycogen** is similar to amylopectin but is even more branched due to glycosidic bonds forming between OH groups on C1 and C4 but also C1 and C6. Glycogen can form **granules** in cells and act as a carbohydrate/energy store. The branches in amylopectin and glycogen make them better for the release of glucose. This is because there are more 'ends' where glycosidic bonds can be hydrolysed and glucose released, which can be used in respiration to produce ATP. The structures of these molecules are shown in the diagram. **Cellulose** **Cellulose** is a complex carbohydrate made of a polymer of *β-glucose* molecules. The β-1,4 glycosidic linkages result in the −CH*X*2 OH groups being on **opposite sides** of the chain of adjacent glucose molecules. Within a cellulose chain, adjacent glucose molecules are rotated 180° relative to each other. This means that OH groups are aligned and a water molecule can be removed to form a glycosidic bond. Therefore, hydrogen bonds do not form between glucose molecules within the same chain, but between glucose molecules in different chains. The hydrogen bonds form **cross-linkages** which hold the chains together. This makes cellulose form into long threads called **microfibrils**. Cellulose is completely **insoluble** and the microfibrils are laid down in overlapping layers in plant cell walls. Cellulose is called a **structural polysaccharide**. It is very difficult to digest because of the very high numbers of hydrogen bonds between the chains of beta glucose. This also gives cellulose very high **tensile strength**; it is difficult to break when stretched. This means that cells with cellulose in their cell wall are more resistant to osmotic lysis (they are not likely to burst because cellulose stops too much water entering the cell). **Chitin** Chitin is found in the cell walls of fungi and in the exoskeletons of insects. It is not a true polysaccharide as it contains the element **nitrogen** -- it is called a **heteropolysaccharide**. It has a similar structure and function as cellulose but because it contains side groups containing N, more hydrogen bonds can form. Chitin microfibrils, therefore, have greater tensile strength than those of cellulose. The diagram shows two of the monosaccharides (N-acetyl glucosamine) found in chitin joined together by a β-glycosidic bond. Lipids are organic compounds made of carbon, hydrogen and oxygen. All lipids contain a high proportion of CH*X*2 groups. Phospholipids also contain **phosphorus**. All lipids have a **low solubility in water** but a high solubility in organic solvents (e.g., ethanol, tetrachloromethane). **Triglycerides** are molecules that form fats or oils depending on the size of the molecule. The more carbon atoms, the higher the melting point because the intermolecular forces are stronger and more energy is required to overcome them. Each triglyceride is made from **glycerol** combined with **three fatty acids** through a **condensation reaction** with the release of **three** molecules of water. The fatty acids bind to the glycerol by means of **ester bonds**. Triglycerides are broken down in a **hydrolysis** reaction with the chemical insertion of **three** molecules of water. The diagram shows how triglycerides are formed and are broken down. The carbon chain in fatty acids is often represented by the letter **R**, a *variable* group containing a chain of between 4 and 24 carbon atoms. Lipids with long hydrocarbon chain fatty acids are more likely to be solid at room temperature -- these are **fats**. Those with short hydrocarbon chains form **oils** which are liquid at room temperature. This is because there are weak forces of attraction between the fatty acid chains -- the longer the carbon chain, the greater the force of attraction, so the higher the melting point. Some examples of different fatty acids and their melting points are shown below. **Name and formula of fatty acid** **Melting point / °C** ------------------------------------------- ------------------------ Lauric acid CH*X*3 (CH*X*2 )*X*10 COOH 45 Palmitic acid CH*X*3 (CH*X*2 )*X*14 COOH 63 Arachidic acid CH*X*3 (CH*X*2 )*X*18 COOH 76 **Functions of triglycerides** Triglycerides are efficient **energy storage** molecules. They are more efficient than carbohydrates: - 1 g fat provides about 38 kJ energy - 1 g carbohydrate provides about 17 kJ energy. Because of this, fats and oils are the preferred energy storage molecule in animals and many seeds (lipids store twice as much energy per gram than carbohydrates). Triglycerides are also good **thermal insulators** and provide **mechanical protection** for delicate organs. Because fats are less dense than water, they are used to provide **buoyancy** for many aquatic animals. Some animals spread oil onto their fur or feathers because this makes them **waterproof**. This is because fats are **hydrophobic** and **repel** water. **Saturated fatty acids** Not only the length of fatty acids can change. They can also be **saturated**, with only **single** bonds between carbon atoms. They contain the maximum number of hydrogen atoms. Lipids containing only saturated fatty acids generally form **fats** at room temperature. This is because the fatty acid tails are straight and can pack closely together. Stronger forces of attraction can form which means more energy is needed to break the bonds and melt the fat -- the melting point is higher. **Unsaturated fatty acids** Fatty acids can also be **unsaturated**, with one or more **double** bonds between *carbon* atoms. They do not contain the maximum number of hydrogen atoms. For each carbon-carbon double bond, the fatty acid will contain two fewer hydrogen atoms. Lipids containing unsaturated fatty acids are usually **oils** at room temperature. The double bonds make the fatty acid tails less straight (they kink) so they cannot pack as closely together. The forces of attraction between the fatty acids are weaker, so less energy is needed to break the bonds and melt the fat -- they have a lower melting point. **Phospholipids** are essential components of cell membranes. Each phospholipid molecule contains a molecule of **glycerol** and: - a **phosphate head** which contains a phosphate ion -- *hydrophilic* - *two* **fatty acid** side chains -- *hydrophobic*. **Functions of phospholipids** The different properties of the phosphate heads and the fatty acids affect how easily different molecules can cross a cell membrane (see Core Concepts 3: cell membranes and transport). If phospholipids are poured into water, the molecules arrange themselves in a single layer. But in cell membranes, phospholipids form a **bilayer**. - The **hydrophilic** phosphate groups are *attracted to water molecules* in the cytoplasm and outside the cell. - The **hydrophobic** tails are *repelled by water molecules* and 'hide' from water in the cytoplasm and outside the cell. Phospholipids can contain saturated and/or unsaturated fatty acids. This also affects the *fluidity* of the membranes. - Those where only saturated fatty acids are present are the *least fluid*. - Those where only unsaturated fatty acids are present are the *most fluid*. The fluidity of the membrane affects how easy it is for the cell membrane to move. A high intake of fat by humans, notably saturated fats, is a contributory factor in heart disease. It raises the low-density lipoprotein (LDL) cholesterol level, which increases the incidence of atheromas in coronary arteries (and in other arteries). This leads to blockages and eventually, heart disease. The table summarises the impacts of different types of dietary fat on human health. **POLYUNSATURATED FAT** **MONOUNSATURATED FAT** ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- An essential fat that we must get from food because our bodies cannot produce it. It lowers LDL (bad cholesterol). **Found in:** most cooking oils, pumpkin seeds, pine nuts, sesame seeds, fatty fish. **Also known as:** omega-3 and omega-6 fatty acids. Considered a healthy fat: it lowers LDL (bad cholesterol) and maintains HDL (good cholesterol). **Found in**: olive oil, avocado and avocado oil, most nuts and nut butters. **SATURATED FAT** **TRANS FAT** Increases total cholesterol and LDL (bad cholesterol). Best to consume in moderation. **Found in**: red meat, whole milk, cheese, coconut, butter, processed meat, many baked goods, and deep fried foods. A by-product of processing healthier fats to give them a longer shelf life. Raises your LDL (bad cholesterol) and lowers your HDL (good cholesterol). Intake should be limited. **Also known as**: partially hydrogenated oil LDL = Low density lipoprotein HDL = High density lipoprotein Proteins are **polymers** made up of about 20 *naturally occurring* subunits called **amino acids**. Each amino acid has a central carbon atom with four different functional groups attached: - the **amino/-NH~2~** group, which has basic properties and can **gain** a H^+^ in acidic conditions to form an -NH~3~^+^group - the **carboxylic acid/-COOH** group, which has acidic properties and can **lose** a H+ in alkaline conditions to form a -COO^-^ group - an atom of **hydrogen, H** - a **variable group, R**. The diagram shows the general structure of an amino acid and the R groups of the 20 common amino acids found in proteins. It also indicates how the R groups affect the properties of the amino acids (which will also affect the properties of any protein). IMPORTANT: you don't have to learn these but it's useful to realise how the R group can change the property of an amino acid and protein. A highly important R group is found in the amino acid **cysteine** where R = -CH~2~-SH~2~ The -SH group of one cysteine can form a covalent bond with the -SH group of another cysteine. This bond is called a disulfide bridge and plays an important role in maintaining the 3D structure of proteins. Amino acids can **polymerise** through a **condensation** reaction to give **dipeptides** and **polypeptides**. The linkage between two amino acids is called the **peptide bond**. Peptide bonds form between the carboxyl group of one amino acid and the amino group of another. The diagram shows the formation of a peptide bond through a condensation reaction and the breaking of a peptide bond through a hydrolysis reaction. It is important to realise that two **different** dipeptides can be formed from two different amino acids. This means that the dipeptides can have different properties because of the arrangement of the amino acids on either side of the peptide bond. In the example in the diagram below, the amino acid at the NH~2~ end -- the N terminal -- will have a basic group at the end, while the amino acid at the COOH end -- the C terminal -- will have an acidic group. This can affect the charges on the amino acid and therefore the properties of the dipeptide. This is also shown in the diagram. A chain of amino acids is called a **polypeptide** -- a series of peptide bonds holding amino acids together. The diagram shows a **pentapeptide** -- a chain of five amino acids. If the order of the amino acids changed, this would produce different pentapeptides, each with different properties. The sequence or order of amino acids in a polypeptide affects the organisation of the molecule as it is processed to form a functional protein. There are **four** levels of protein structure or organisation. **Primary structure** Each protein and polypeptide has a specific **primary** structure which is based on: - **which** amino acids are present - the **number** of each type of amino acid present - the **sequence** of amino acids in the polypeptide chain. If you are asked to define the primary structure, then you should say 'the sequence of amino acids in a polypeptide chain'. **Secondary structure** Each chain of amino acids contains many **polar** groups: - the amino group −N−H*Xδ*+ - the carboxylic acid group −C=O*Xδ*−. Because opposite charges attract, the −N−H*Xδ*+ and −C=O*Xδ*− groups attract each other, forming **hydrogen bonds**. These make the amino acid chain **fold** and **twist** into a new shape. This new shape is called the **secondary structure** of the protein. *This folding does not involve the R groups.* The two most common types of secondary structure are: - **alpha helix** - **beta pleated sheet**. The diagram shows the hydrogen bonding in these types of secondary protein structure and also how they are represented in **ribbon diagrams** which try to show the 3D structure of a protein. Proteins with a secondary structure play important **structural** roles in organisms. - The **alpha helix** gives rise to **fibrous** proteins where several strands of alpha helices can be coiled together to give a rope-like arrangement. These are insoluble in water and have a **structural** function in organisms. - E.g., **α-keratin** in wool\ **collagen** in skin and blood vessels. - **Beta pleated sheets** form layers of protein.\ E.g., **fibroin** in silk. **Tertiary structure** The **tertiary structure** of a protein or polypeptide depends on the properties of the **R groups**. The different chemical natures of the variable groups make them interact with each other and form several types of bond. - **Ionic** bonds are formed from charged variable groups and can interact with water, which helps a protein to dissolve. - **Covalent** bonds are formed from variable groups containing **sulfur** atoms -- two of these can bond together to form a **disulfide bridge**. As they are covalent bonds, disulfide bridges are strong and more difficult to break. A higher temperature or more extreme pH would be needed to break these bonds. - Additional **hydrogen** bonds can also form between polar variable groups. - **Hydrophobic** interactions take place when the variable groups are **non-polar**. They are **repelled** by water and are usually found on the inside of the protein as far away from water as possible; a protein rich in non-polar side groups will be less soluble in water. The additional folding of the protein gives rise to a compact, **globular**, **three-dimensional** shape that makes the protein soluble in water -- charged groups on the outside and hydrophobic groups on the inside. The tertiary structure gives globular proteins a specific 3D shape which gives the protein its function. Many globular proteins have a **metabolic** function in organisms: - **enzymes** -- *active sites* to bind to a *substrate* - **antibodies** -- sites for binding to *antigens* - **hormones** -- sites for binding to *specific receptors*. The diagram shows some of the different bonds that can form between side groups. **Quaternary structure** Some proteins are made of two or more polypeptide chains, combined to form the fully functional protein. Each of the polypeptide chains has a primary, secondary and sometimes tertiary structure. This arrangement of several polypeptides is called the **quaternary structure** of a protein. In some proteins with a quaternary structure, bonds called **disulfide bridges** bond the polypeptide chains together, for example: - **insulin** - **haemoglobin** - **immunoglobulins**. These are all **globular** proteins which have a **metabolic** function in the body. In other proteins, **hydrogen bonds** bind the polypeptides together for example: - collagen - silk. These are **fibrous** proteins which have a **structural** role in the body. The high number of hydrogen bonds between the polypeptide chains in collagen stops the chains sliding past each other and makes collagen strong. Examples of proteins with quaternary structure are shown in the diagram below. Enzymes nzymes are globular proteins which increase the rate of reaction by lowering the activation energy of the reaction they catalyse. Active site is the area of the enzyme where the reaction with the substrate takes place. Enzymes are specific to substrates they bind to meaning that only one type of substrate fits into the active site of the enzyme. When the enzyme and substrate form a complex, the structure of the enzyme is altered so that the active site of the enzyme fits around the substrate. This is called the induced fit model. The lock and key model of enzyme activity is based on the idea that the substrate fits into the enzyme the way a key fits into the lock due to the complementarity in shape between the two structures. Enzymes can be intracellular and catalyse reactions inside of cells, for instance ATP synthase, DNA helicase which unwinds the helix, DNA polymerase involved in catalysing the formation of phosphodiester bonds as well as lysosome which carries hydrolytic enzymes. Examples of extracellular enzymes are digestive enzymes secreted by the cell. Enzymes can be immobilised for the use in industrial processes with the purpose of making the enzyme more stable and reusable. Factors affecting the rate of enzyme-controlled reactions: Enzyme concentration -- the rate of reaction increases as enzyme concentration increases as there are more active sites for substrates to bind to, however increasing the enzyme concentration beyond a certain point has no effect on the rate of reaction as there are more active sites than substrates so substrate concentration becomes the limiting factor. Substrate concentration -- as concentration of substrate increases, rate of reaction increases as more enzyme-substrate complexes are formed. However, beyond a certain point the rate of reaction no longer increases as enzyme concentration becomes the limiting factor. Temperature -- rate of reaction increases up to the optimum temperature which is the temperature enzymes work best at, rate of reaction decreases beyond the optimum temperature. pH -- in the case where the pH is more acidic than the optimum pH, H+ ions disrupt the enzyme-substrate binding and decrease the rate of reaction. In the case where the pH is more alkaline than the optimum, the OH- ions disrupt the binding, also leading to a decrease in product formation. www.pmt.education Inhibitors An inhibitor is a substance which slows down or stops a reaction by affecting the binding of substrate to the enzymes. Inhibitors can either be reversible and irreversible. Examples of irreversible inhibitors include heavy metal ions such as mercury and silver which cause disulphide bonds within the protein structure to break, as a result causing the shape of the active site to change, thus affecting protein activity. Other examples include cyanide which is a nerve gas that covalently binds to the active site, therefore preventing the binding of the substrate. Reversible inhibitors bind to the active site through hydrogen bonds and weak ionic interactions therefore they do not bind permanently. Reversible inhibitors can either be competitive or non-competitive. Competitive inhibitors are similar in structure to the substrate molecule therefore they bind to the active site of the enzyme, decreasing its activity as they compete with substrate for the enzyme. The amount of product formed remains the same, however the rate at which product formation occurs decreases. The higher the concentration of competitive inhibitor the lower the reaction rate. Increasing the substrate reverses the effect of competitive inhibitors by outcompeting them. Non-competitive inhibitor does not bind to the active site, it binds at another site on the enzyme known as the allosteric site. Binding of the non-competitive inhibitors changes the shape of the active site therefore preventing the binding of the substrate. As a result, the enzyme-substrate complex does not form. Increasing the concentration of substrate has no effect on non-competitive inhibition. Many drugs are inhibitors. Examples include penicillin which is used to fight bacterial infections, it is an inhibitor of enzyme transpeptidase which plays an important role in cell wall formation. Other examples include Ritonavir which is an antiretroviral drug used to treat HIV which inhibits HIV protease which is responsible for assembly of new viral particles and spread of infection. Digitalis, a chemical extracted from foxglove which can be used to treat arrhythmia is a non-competitive inhibitor which binds to an enzyme involved in restricting contraction of the cardiac muscle. Gas exchange in animals Organisms need to exchange materials with their environment. Every cell in a living organism (its volume) requires a supply of oxygen and glucose sufficient to meet its respiratory demands. Cells also need water. As cells metabolise, they produce waste products like carbon dioxide, nitrogenous waste and water. These need to be eliminated from the volume of the organism. The cell membrane is where these materials are moved into or out of the cell. Gases and non-polar substances cross the cell membrane by simple diffusion, through the phospholipid bilayer. Polar and charged substances cross the membrane through specific protein carriers or channels by facilitated diffusion. Water crosses by osmosis. Substances moving from low concentration to high are transported by active transport using specific protein carriers and ATP. Larger particles and some liquids move by endo- or exocytosis. Exchange of materials takes place over the surface area of the cell or organism. This needs to be large enough to supply every cell in the volume with the materials to fulfil its metabolic needs. As organisms increase in size, they have more cells and more volume and a higher requirement for oxygen. As size increases volume increases more than surface area. A way of expressing this, so that organisms of different sizes can be compared, is to divide the surface area by the volume. The result is the surface area to volume ratio, X:1. As organisms increase in size, their surface area to volume ratio gets smaller. The rate of diffusion depends on surface area, diffusion path and concentration gradient. The larger the surface area the more membrane there is and the higher the rate of diffusion. Folded surfaces (microvilli) have an even higher surface area. The smaller the diffusion path the less distance there is to travel and the higher the rate of diffusion. Flatter cells and having only a single layer of cells shorten diffusion paths. The higher the concentration gradient the faster the rate of diffusion. Ventilation mechanisms bring supplies of fresh oxygen and remove carbon dioxide. Circulation systems carry carbon dioxide away from cells and bring fresh oxygen to them. The use of oxygen in respiration and production of carbon dioxide in cells maintains concentration gradients. Movement of aquatic organisms can increase the flow of oxygenated water and help maintain concentration gradients. Turbulence in water increases oxygenation and therefore the concentration gradient. Unicellular organisms like *Amoeba* have a high surface area to volume ratio. It is also a small distance to the centre of the cell, so diffusion paths are short. Thus, by diffusion across their cell membranes, unicellular organisms can gain sufficient oxygen and glucose fast enough to sustain their metabolism and do not need specialised gas exchange surfaces. Concentration gradients depend on oxygen use and carbon dioxide production only. Flatworms are multicellular organisms with a smaller surface area to volume ratio than *Amoeba*. They have a flattened body which shortens the diffusion path to the centre of the body. Being long and flat gives them a high surface area to volume ratio. Flatworms have no specialised gas exchange surface; their shape enables them to gain sufficient oxygen by diffusion fast enough to meet their needs over their surface. Earthworms are terrestrial animals that are multicellular. The long cylindrical shape of an earthworm gives it a high surface area to volume ratio. The diffusion distance, from the surface to centre of the animal, is too large for diffusion alone to supply oxygen at a sufficient rate to sustain its metabolic requirements. Earthworms have a circulatory system to maintain diffusion gradients. The blood vessels are close to the external surface of the organism so that the diffusion path from the surface to the blood is short. As the blood flows in blood vessels, earthworms have a closed circulatory system. The blood contains haemoglobin that has a high affinity for oxygen and transports it to the tissues. Earthworms do not have a specialised gas exchange surface; gases diffuse to the blood across the surface of the animal. On the model in the image, the blood vessels are coloured dark red, and you can see the five pseudohearts that push the blood around and the dorsal and ventral blood vessels. Watch the two videos and make notes on anything you think will help. Amphibia are larger multicellular animals with aquatic and terrestrial modes of life. Amphibia must return to water to breed and their tadpoles are aquatic using gills for gas exchange. The gas exchange surface of an adult amphibian is its thin moist skin. A closed circulatory system containing blood with haemoglobin transports oxygen to the tissues. At rest, this is sufficient to supply the needs of the animal. During periods of activity, amphibia also use primitive lungs. These lungs are not highly folded like the lungs of mammals but do increase the surface area for gas exchange. Their lungs are internal which reduces water loss and heat loss. Larger multicellular animals have specialised gas exchange surfaces which overcome the problems with a low surface area to volume ratio and long diffusion distances. Generally, these gas exchange surfaces must have: - a large surface area - a short diffusion path - ventilation mechanisms for maintaining a concentration gradient - permeability to gases - moist surfaces so oxygen can dissolve in the water and diffuse across them. Some animals have a circulatory system to maintain concentration gradients by bringing oxygenated blood to tissues and removing carbon dioxide. Haemoglobin has a high affinity for oxygen and absorbs and transports oxygen. The surface of a fish is covered in scales which are impermeable to water and gases. Resultantly, fish exchange gases through gills, more precisely the gill filaments and the gill plates. The gills are covered by an operculum, which opens to let stale water (with high carbon dioxide) out. The operculum closes to increase the pressure within the gill cavity during ventilation movements. Each gill cavity contains four gills supported on bony gill arches. The gills are feathery structures consisting of many gill filaments. The filaments are covered with many lamellae/gill plates at right angles to the filaments which increases the large surface area. The lamellae are very thin and are close to the capillaries, providing a short diffusion path for oxygen and carbon dioxide. On the other side of the gill arches are the gill rakers, which filter large particles out of the water preventing damage to the delicate gill filaments. The gill filaments have a rich blood supply; the blood contains haemoglobin. Download the practical schedule for the fish head dissection below. If you are unable to complete this at school, fish heads are often free from fishmongers, and trout and mackerel are usually sold with their heads on. A good pair of kitchen scissors will suffice for the dissection and a teaspoon or wooden skewer will help you to spot the structures you need to. The main hazard in any dissection is that the instruments you use are sharp and there is a risk of cutting yourself. You should control the risk by cutting away from yourself. Make sure you can recognise the structures from the photograph in the practical schedule. More active fish with a higher oxygen requirement may have thinner gill filament, more gill filaments or more gill plates. [Download file](https://resource.download.wjec.co.uk/vtc/2021-22/ebl21-22_9-8/Downloads/TG%2014%20Dissection%20of%20fish%20head%20to%20show%20the%20gas%20exchange%20system.pdf) In cartilaginous fish, such as sharks, blood and water flow in the same direction across the gill plates. This flow is called concurrent or parallel flow. Blood with low oxygen coming from the body is in contact with water with high oxygen coming in through the mouth. Oxygen diffuses into the blood until equilibrium is reached. In bony fish, blood and water flow in opposite directions across the gill plates. This flow is called countercurrent flow. Blood with high oxygen leaving the gill is in contact with water entering the gill from the buccal cavity, which has a higher oxygen content than the blood. Blood entering the gill filament has low oxygen concentration but is meeting water as it leaves having had oxygen removed. Despite this, the water still has a higher oxygen concentration than the blood. The water has a higher oxygen concentration than the blood across the whole distance of the gill plate, so equilibrium is never reached, and the concentration gradient is maintained over the entire gill. This means that diffusion occurs along the entire length of the gill and more oxygen is absorbed. The gas exchange system in mammals consists of the following: **Larynx**: this is the 'voice box' and contains the vocal cords enabling sounds to be produced. **Trachea**: a pipe that connects the lungs to the pharynx; it has C-shaped rings of cartilage that prevent the trachea collapsing when pressures in the lungs are negative. **Bronchi**: two bronchi branch off the lower end of the trachea delivering air to each lung. Rings of cartilage prevent the bronchi collapsing. **Bronchioles**: smaller branches from the bronchi delivering air to all parts of the lung; muscle permits constriction to control the flow of air in and out of the alveoli. **Alveoli**: the site of gas exchange, these consist of sacs of air surrounded by flattened (squamous) epithelia. The large number of alveoli give a massive surface area to the lungs and the flattened epithelial cells give a short diffusion path. The alveoli are surrounded by capillaries which maintain a concentration gradient. The blood contains haemoglobin which transports oxygen away from the gas exchange surface. Carbon dioxide diffuses from the plasma into the alveoli to be excreted. **Pleural membranes**: these surround the lungs and secrete pleural fluid. The pleural membranes are involved in negative pressure breathing. **Ribs**: protect the heart and lungs. **Intercostal muscles**: these sit between the ribs, contraction of these muscles raises the rib cage in ventilation. **Diaphragm**: a muscle that separates the thorax and abdomen, contraction of this muscle pulls the diaphragm down in ventilation. Goblet cells in the ciliated epithelial layer produce mucus which trap particles in inspired air. Cilia sweep the mucus upwards to the pharynx so that they can't reach the lungs. The rings of cartilage prevent the trachea collapsing during inspiration. The rings are incomplete which allows the trachea to collapse slightly when food passes down the oesophagus; this increases the size of the oesophagus, so it is easier for food to pass down. The smooth muscle enables the trachea to reduce its diameter making coughs more forceful, which is useful when trying to expel material. Many alveoli give a large surface area for gas exchange. The alveoli have a single layer of squamous epithelial cells giving a short diffusion pathway. The dense network of capillaries is close to the alveoli, giving a short diffusion pathway and large surface area and circulation of the blood maintains a diffusion gradient. The alveoli are moist so that gases can dissolve. Surfactant in the alveoli reduces the surface tension of the water and prevents the alveoli collapsing. Premature babies do not produce surfactant and can be treated with surfactant so that their alveoli remain open. Ventilation in mammals is by negative pressure. You will recall that fish have a positive pressure ventilation mechanism. Ventilation movements bring oxygenated air to the lungs and remove carbon dioxide rich air from the lungs; this maintains a concentration gradient between the blood and alveoli. Inspiration (breathing in): - The intercostal muscles contract, pulling the rib cage outwards and upwards. - The diaphragm contracts, pulling the diaphragm downwards and flattening it. - The external pleural membrane is pulled outwards. - The pressure in the pleural cavity decreases. - The inner pleural membrane moves outwards (to the negative pressure) pulling on the lungs and expanding the alveoli. - The volume of the lungs increases, and the pressure decreases. - The alveolar pressure falls below that of the atmosphere and air flows in through the trachea, bronchi and bronchioles and passes into the delicate alveoli. In expiration (breathing out): - The muscles relax and the opposite happens. Insects have a high surface area to volume ratio making them susceptible to water loss and desiccation. An adaptation for their terrestrial mode of life is that they are covered in a chitin exoskeleton which has a layer of waterproof wax on it. This means that they cannot exchange gases over their surface as the wax is not permeable to gases. Insects have holes in their exoskeleton called spiracles. The spiracles lead to tracheae -- a system of chitin lined tubes. The chitin supports the tracheae and prevents them collapsing. The tracheae terminate in tracheoles which are close to the cells of the insect. The tracheoles are the gas exchange surface. Insects have an open circulatory system; the 'blood' is in direct contact with the cells and is not in blood vessels. Insects do not have haemoglobin to transport oxygen. The oxygen is delivered direct to the cells through the tracheal system. This system depends on diffusion alone through the tracheal system from the environment to the cells. This relies on diffusion gradients which are maintained by respiration in the cells. Oxygen is used in the cells, making the oxygen concentration low, so oxygen diffuses towards the cells. Carbon dioxide is produced by respiration in the cells which makes the carbon dioxide concentration high, so carbon dioxide diffuses away from the cells towards the spiracles. Air sacs in the insect act as a store of oxygenated air. Spiracles are a row of holes down each side of the insect. Insects have a body divided into three distinct sections, a head, thorax and abdomen. Only the thorax and abdomen have spiracles. In most insects the spiracles have valves so that they can close to reduce water loss. In order to exchange gases, the spiracles must be open. A build-up of carbon dioxide in the tracheae stimulates the opening of the spiracles so that the carbon dioxide is released, and oxygen rich air diffuses in. The drop in carbon dioxide level in the tracheae stimulates the spiracles to close reducing water loss. Spiracular fluttering describes the rapid partial opening and closing of spiracles to allow gases to enter and leave. This is punctuated by full opening of spiracles. Ventilation in insects is accomplished by muscular movements of the abdomen. To bring fresh air into the insect the abdomen expands, lowering the pressure inside. The abdominal spiracles are closed and the spiracles on the thorax are open. The lower pressure in the abdomen pulls the air in through the thoracic spiracles. To expel carbon dioxide rich air, the abdomen contracts lowering the volume and increasing the pressure. The thoracic spiracles close and the abdominal spiracles open. The stale air is forced out of the open spiracles. Insect flight muscle is a highly active tissue. The cells contain many mitochondria which utilise oxygen in aerobic respiration and release ATP to be used in muscle contraction. The tracheoles in flight muscle penetrate the cells. At rest the tracheole ends are fluid-filled. During flight, the available oxygen is utilised rapidly, and some anaerobic respiration takes place. Anaerobic respiration produces lactic acid. The lactic acid lowers the water potential of the muscle cells and the water leaves the tracheoles by osmosis. The result of this is that there is more air in contact with the muscle cells, raising the rate of diffusion of oxygen into the cells. Human impact (conservation, deforestation, overfishing, planetary boundries) Extinction **results in a loss of species** and a decrease in biodiversity. Natural selection can be a reason for extinction. During the process of evolution, various species have been replaced by others. An **endangered species** is a species that is seriously at risk of extinction. **The possible reasons for species becoming endangered due to human impact include:** - Destroying habitats -- e.g. removing hedges, draining wetlands, deforestation. - Pollution of the environment e.g. PCBs (polychlorinated biphenyls) oil and pesticides. - Introduction of alien species to an ecosystem. These species may out-compete native species for food and space. In addition, they might have no natural predators or grazers to control the population. They may also carry diseases that affect native populations. - Monoculture -- growing large numbers of the same, genetically identical individuals in a given area. - Building roads, houses, factories. - Unsustainable harvesting e.g. over-hunting / over-fishing. **Conservation** involves the creation, management and protection of habitats. Ideally, conservation maintains the biosphere and enhances biodiversity locally. The conservation of existing gene pools in the wild and in captivity is vital for maintaining biodiversity for the future. **There are many ways in which conservation may be achieved:** - Habitat protection by nature reserves and SSSI (sites of special scientific interest) e.g. coral reefs. - International cooperation between governments and organisations e.g. to restrict trade in animal parts such as ivory, or to ensure that the international trade in animal and plant specimens does not threaten their survival (CITES). - Restricting activities that threaten an endangered species e.g. whaling. - Legislation to prevent overfishing, poaching, collecting birds\' eggs, and picking wild flowers. - Breeding programmes by zoos and botanic gardens. - Sperm banks and seed stores. - Reintroduction programmes such as the Red Kite in Mid Wales. - Pollution control. When a species is conserved, the gene pool of that species is also conserved. In the wild, it is important to maintain the genetic diversity of the species. Genetic diversity is critical if the species is to survive changing environments as natural selection is dependent on variation in the species. In addition, some organisms may have alleles that are useful to humans. Our domestic animals and plants all have wild relatives that may have useful alleles that could be bred back into the domestic varieties and confer e.g. disease resistance. This is the point of rare breeds, frozen zoos, and sperm and seed banks. Furthermore, many plants have medicinal properties and if they become extinct before investigation that resource is lost. Captive breeding programs have a moral obligation to maintain the genetic diversity of the stocks of captive animals, to ensure the diversity of the species. With an ever-increasing population to feed, there is a constant tension between the need to produce more food and the need to conserve the environment and biodiversity. Agriculture is the means of producing food for human consumption in order to meet demand. To meet this growing demand, agriculturalists have: - created larger fields by removing hedges - cultivated monocultures (growing a single type of crop in a field) - increased their use of fertilisers and pesticide. However, these practices can have a negative effect on the environment and biodiversity. **Hedges** provide valuable habitats for wildlife, including some pest species and their predators. They also provide "wildlife corridors" allowing mobile species to travel to different areas to disperse species and find mates. **Monocultures** allow farmers to grow crops that are easy to harvest mechanically. They can also be guaranteed to yield disease-free seeds. However, if pest species invade a monoculture, they can increase in numbers very rapidly and a monoculture is less likely to sustain a variety of the pests' predators which might keep their numbers under control. **Fertilisers** can cause eutrophication of nearby water bodies as any excess will leach into rivers. **Pesticides** harm beneficial species as well as pests and can cause them to decline in numbers. Throughout human history, forests have been exploited, whether for timber, firewood, wood building or cash crops. Land has been cleared of trees in order to grow food and to rear livestock. One of the concerns that environmentalists have is the rate at which **deforestation** is happening, particularly in the tropics. Large areas of woodland are cleared and burnt using large machinery which compact the ground and necessitates road building. Succession is less likely to occur in these areas, leading to soil erosion. This means that tree seedlings cannot become established and the area becomes a desert -- the name for this process is **desertification**. Deforestation can lead to: - the destruction of natural habitats and niches - a decrease in native biodiversity - an increase in soil erosion leads to an increase in nutrient loss - succession from cleared land doesn't occur because the top soil has been lost - increased sediment deposits in waterways - loss of valuable plant materials which could have potential medicinal uses - contributes to global warming (more carbon dioxide in the atmosphere because there are fewer trees to absorb it). **Possible conservation methods** Sustainable management means that timber can still be extracted without destroying the forest. This means that succession can happen after the trees are removed, and therefore the forest can regenerate for future harvesting. In Britain, the traditional method is coppicing -- woodland is divided into different areas to be cut down in rotation. Trees are cut to the stumps and the wood re-grows from the stump. The trees produce long straight stems which can be harvested. Introduction of protected areas and replanting of native species within woodlands can also help preserve species and also promote biodiversity. Fish stocks (populations) are renewable, that is, individuals that are lost through deaths are replaced by births. The populations can be harvested (fished) indefinitely -- because of this, fish is a sustainable resource. However, **overfishing** can threaten the sustainability of fish populations. Overfishing is defined as the level of fishing where **increased effort results in a declining catch**. Too much fishing means that numbers within a population cannot be maintained, leading to a decline in the fish stocks. **Reducing the impact of overfishing:** - **Fishing quotas** -- these limit how many members of any one species can be caught. Heavy fines are imposed for exceeding quotas. - Reducing the size of **fishing fleets** - this means that fewer boats are out catching fish. - **Restricting seasons for fishing** -- by banning people from fishing during breeding seasons, the fish stock should have enough time to replenish. - **Restricting mesh sizes for fishing nets** -- smaller mesh sizes can catch more fish because they can catch both small and large fish. By having a minimum mesh size, larger fish will be caught but smaller fish, who are often juveniles, will be able to escape from the nets and go on to breed. - Banning fishing from some zones (**exclusion zones**) altogether. In exclusion zones, the fish population will remain at sustainable levels. - **Fish farming** -- this involves isolating an area of sea for the purpose of breeding and growing fish in managed conditions. As with terrestrial farms, the animals are fed, treated with chemicals (to keep them pest and disease-free) and then harvested. But fish farming can lead to problems in aquatic ecosystems: - Provision of additional nutrients can lead to **excess nutrients in the area leaking out** and leading to eutrophication. - Overuse of antibiotics can lead to resistance in pathogenic bacteria. - Non-specific pesticides can leak out, affecting marine food chains. - The fish in the netted-off area can be overcrowded. Because of this, diseases and pests can spread easily. - Fish in captivity are sometimes genetically modified for fast growth rates and larger sizes. This could have serious consequences should the fish escape and breed with wild stocks or compete with them for food. To try and prevent the breeding problem, many genetically modified fish have been engineered to have 3 complete sets of chromosomes (trisomy). **Environmental monitoring** describes physical and biological measurements that are made over a period of time. Physical measurements might include pH, drainage or water flow. Environmental impact assessments are required before work can start on construction projects such as rail-lines, roads, wind farms, tidal barrages, airports and buildings. This process of monitoring and assessment helps political bodies make decisions based on sound scientific data about the impact of projects on the environment and to consider alternatives where necessary. Environmental monitoring is also required to monitor the effects, assess the effectiveness, and measure the environmental impact of conservation and re-introduction programmes. Some aspects that are monitored routinely are: - Chemicals -- this includes pH, carbon dioxide, nitrates and ammonium - Biotic -- animals and plants, especially those that are sensitive to change. A good example is the monitoring of brown trout and salmon as indicators or water quality and oxygenation - Radiation - Microbes -- especially for areas that are used for recreation (e.g. lakes and rivers) which could be unsafe should the levels be dangerous. Monitoring may involve using transects or random sampling of areas. Often the experiments need repeating over time and at different seasons to assess the impact. 1. **Biosphere integrity** describes biodiversity loss and extinctions. The main cause for this is increased demand for food, water and natural resources. Habitats are being lost rapidly, e.g. coral reef bleaching caused by ocean acidification and rising temperatures. This boundary has already been exceeded. 2. **Climate change:** the level of CO~2~ has risen dramatically and continues to do so. This boundary has been crossed and scientists believe that the loss of polar sea-ice could be irreversible. This could push global temperatures and sea levels up. So too could the destruction of rainforest and weakened carbon sinks in tundra and oceans accelerate global warming and climate change. 3. **Chemical pollution and novel entities:** the emission of toxic and long-lived substances, such as heavy metals, radioactive materials and synthetic organic pollutants. These can cause reduced fertility and genetic damage, e.g. DDT dramatically reduced bird populations. These have yet to have a quantified boundary. 4. **Ozone depletion:** the ozone layer, a region of the stratosphere which shields the earth from UV radiation from the sun, has depleted in recent decades due to chemical pollution, e.g. CFCs from refrigerants and aerosols. Actions taken as a result of the Montreal Protocol, an international treaty signed in 1987, mean that this boundary has not been exceeded. 5. **Aerosol loading:** aerosols are atmospheric pollutants. Their effects in the atmosphere are complex and they have yet to be quantified. 6. **Ocean acidification:** this is owing to increased CO~2~ dissolving into oceans and forming carbonic acid. Organisms with calcium carbonate shells, like corals and molluscs can't make shells in acid water. This has a knock-on effect on food chains and webs and could drastically reduce fish stocks. This boundary could be avoidable, but is approaching amber. 7. **Biochemical flows:** concern the nitrogen and phosphorous cycles. Both elements are fixed into fertilisers but up-take by plants is limited leading to eutrophication. The production and application of fertilisers continues to be a concern. 8. **Freshwater consumption and the water cycle:** not yet quantified. Nevertheless, globally fresh water is becoming scarce because of modification of water bodies and land use change. Desalination of sea water may ease the situation. 9. **Land system change:** this describes land converted to human use e.g. for agriculture. Globally this is approaching **red**.