Enzyme Kinetics: Lactate Dehydrogenase

Enzyme Kinetics: Lactate Dehydrogenase Question: What scientific question was this part of the practical designed to address? What is the effect of heat stability on two isoforms of the enzyme lactate dehydrogenase (LDH1 and LDH5) after pre-incubation (heat inactivation) of the enzyme samples at different temperatures? Approach: Outline and explain the experimental approach that was used to address the question. In order to determine the effect of the heat inactivation on LDH1 and LDH5, a colorimetric assay was carried out using different reaction mixtures prepared with LDH1 and LDH5. 16 test tubes were prepared each with a specific volume of LDH1 or LDH5. The same concentration of substrate solution containing lactate and NAD+ was added into each of the 16 tubes except for two tubes with the blank solution. Each of the enzyme samples were pre-incubated at 37 °Ãƒ Ã‚ ¡, 44 °Ãƒ Ã‚ ¡, 50 °Ãƒ Ã‚ ¡, 54 °Ãƒ Ã‚ ¡, 58 °Ãƒ Ã‚ ¡, 62 °Ãƒ Ã‚ ¡ and 66 °Ãƒ Ã‚ ¡. The reaction mixtures had to be mixed, incubated for 37 °Ãƒ Ã‚ ¡ and allowed to cool. This gives the lactate dehydrogenase enough time to work on the substrates under an optimum temperature. The lactate dehydrogenase catalyzes a reversible reaction by converting lactate and NAD+ to pyruvate, NADH and H+ or from pyruvate to lactate. The overall effect of the enzyme is that it transfers a hydride ion from lactate to NAD+ or from NADH to pyruvate. (Zhadin N et al 2008). The NADH produced in the LDH catalysed reaction has absorption properties (wavelength) that are greater than that of NAD+ which makes it useful for this experiment. When the colour reagent is added to the reaction solutions, NADH was used to form formazon (blue) according to the principle: Lactate + NAD+ → Pyruvate + NADH + H+ NADH + Phenazine methosulphate + H+ → NAD++ Reduced phenazine methosulphate. Reduced phenazine methosulphate + Tetrazolium → Formazan + Phenazine Methosulphate.The extent to which the enzymes have been heat inactivated will be reflected on the intensity of the formazon produced. The formazon was measured at 520nm in the spectrophotometer against the blank solutions for LDH1 and LDH5. The results obtained allowed us to compare how effective the LDH1 and LDH5 is at different pre-incubation temperatures and helps us to study the heat stability of the isoenzymes. Data: In figure 1 the absorbance begins to decline after around 44  °C in both enzyme isoforms. It is evident LDH5 has a higher absorbance than LDH1 at relatively low temperatures, but at 49 °C there is an overlap between the isoenzymes and after this point LDH5 continues to have a lower absorbance reading than LDH1. This shows the enzyme is losing effectiveness at catalysing the conversion reaction at temperatures higher than 44 °C suggesting this may be the enzyme’s optimum temperature at which it is most effective. With LDH1 there is a steady decrease in the enzyme activity of around 20-30% from 44 °C to 58 °C. After 58 °C there is a sharp drop in the effectiveness of the enzyme by around 60%. The decline may be due to the active site of the enzyme being denatured with exposure to the high pre-incubation temperature. The LDH5 begins with an increasing enzyme activity from 37 °C to 44 °C and after 44 °C there is a greater drop in enzyme activity of around 80%. The sharp drop in enzyme activity occurs much quicker in LDH5 than in LDH1 (figure 2), because there is a rapid loss of effectiveness when the temperature is raised above 50 °C. LDH5 also loses function at a lower temperature compared to LDH1; it becomes inefficient at 58 °C, where the curve remains consistent with a very low enzyme activity (0.31-0.62%). In addition, the LDH1 isoform demonstrates half maximal activity at 11.4 °C greater than that of LDH5 illustrating LDH1 is more tolerant to increasing temperatures. The results have shown that LDH5 has low heat stability and becomes inefficient at a lower temperature than LDH1. Discussion/Conclusions: The lactate dehydrogenase is characterised by its isoenzymes that are tetramers. The tetramers are made up of four monomers; H4 (heart) or M4 (muscle). The monomers are formed by a polypeptide chain of amino acids which consists of an active site. When there is lactate and NAD+ present in the solution, the peptide loop in lactate dehydrogenase blocks the access to the binding site after the substrates have bound; this aids the transition state of the reaction. The difference in heat stability between the two isoenzymes in the experiment can be due to the amino acid sequence of M and H subunits. The amino acid composition of M and H subunits consist of the same amino acids, however the difference is that they have varying numbers of each amino acid. LDH1 has a greater composition of valine, aspartic acid and methionine residues in comparison to LDH5 (Goldberg E.1972).The higher the methionine content in LDH1 means there is more sulphur atoms capable of forming strong covalent disulphide bridges, which can be why LDH1 is more heat stable as more energy is required to denature the isozyme compared to LDH5. Heat inactivation of the isoenzymes can affect the protein structure of lactate dehydrogenase. To form the primary structure, the -COOH group of one amino acid combines with the -NH of another to form a peptide bond. Within proteins there are regions that are arranged into alpha helices and beta pleated sheets. The bonds that hold these together are hydrogen bonds between lone pairs of oxygen and a hydrogen atom. The alpha helix is when the polypeptide is in a spiral form allowing more hydrogen bonds to form between the residues. Similarly, the beta pleated sheet consists of polypeptide chains running parallel or anti-parallel to each other. The way the polypeptide chain is arranged allows more hydrogen bonds to be formed. The two secondary structures fold up differently to form a 3D shape. There are various bonds that holds this shape together: ionic interactions between -COOH and -NH2 groups of different amino acids, hydrogen bonds between the side chains, van der Waals forces wher e fluctuating dipole in one of the groups of the amino acid can create a dipole in another which is used to keep the folded structure together, hydrophobic and disulphide bridges. A quaternary structure is formed by interaction of several polypeptide chains/subunits (LDH1 consists of 4 H subunits). In conclusion, the heat inactivation can interfere with the different interactions between the amino acids that make up the protein structures. The heat inactivation has shown to provide enough energy to overcome some of the covalent bonds to denature the enzymes. Since LDH1 is found to have a negative charge it may be associated with many ionic interactions which needs more heat energy is needed to overcome the strong bonds, hence why it takes a greater temperature (66 °Ãƒ Ã‚ ¡) for LDH1 to lose enzyme activity. The strong bonds holding the LDH1 together means the enzyme is not destroyed at 60 °Ãƒ Ã‚ ¡ however LDH5 is destroyed which is evident by its inability to function adequately after 55 °Ãƒ Ã‚ ¡. Experiment 2: Question: What scientific question was this part of the practical designed to address? How to study the kinetic parameter of different LDH isoenzymes by finding the Vmax and Km values for LDH1 and LDH5. Approach: Outline and explain the experimental approach that was used to address the question. To study the kinetic parameters of different LDH isoenzymes, the experimental procedure and principles where exactly the same when the reaction mixtures were prepared before. However, the LDH isoenzymes were added to different concentrations of substrate solutions and the temperature was constant. A coloured complex is formed using the mechanism in the previous experiment which produces blue formazon. The absorbance of the formazon was measured using the spectrophotometer; the absorbance measured indicates the enzyme activity. The results from this experiment will help to understand the effect of varying substrate concentration has on the enzyme activity and compare this between the two isoenzymes. To determine the Vmax and Km values more accurately the Lineweaver-Burk-plot is used. Data: Table of results for LDH1 and LDH5 LDH1: LDH5: y = 6.2734x + 0.9242 y = 27.641x + 0.1673 Table of results for Vmax and Km in both isoenzymes. Discussion/Conclusions: As the substrate concentration is increased, the formation of the product will also rise but there is a point at which increasing the substrate concentration any further will become a limiting factor. The concentration at which this occurs is greater than 80mM which may decrease the enzyme activity in both LDH1 and LDH5. Since Km and Vmax is greater in LDH5 indicating the affinity of LDH5 for its substrate is much lower so a greater substrate concentration is needed to reach Vmax. The high Vmax for LDH5 means the saturated lactate dehydrogenase is converting more of the substrates into its products at high substrate concentrations. As a result LDH1 will be a more ideal isoenzyme than LDH5. In this experiment we observed the catalysis of lactate dehydrogenase from lactate to pyruvate. The pyruvate produced has the ability to enter the mitochondria, where pyruvate dehydrogenase links the glycolysis metabolic reaction to the citric acid cycle. In a process of pyruvate decarboxylation, the pyruvate is converted into acetyl-CoA, which releases energy by converting NAD+ to NADH and H+. Pyruvate has the potential to either enter the mitochondria to become acetyl-CoA or become lactate. The Cori cycle is involved in the conversion of lactate to pyruvate. When lactate is produced in the muscle, it is released into the bloodstream and transported to the liver. In the liver the lactate is first converted to pyruvate by lactate dehydrogenase and pyruvate is converted to glucose through gluconeogeneis. Lactate dehydrogenase becomes activated when there is a high concentration of its substrates, NAD+ and lactate. For instance when the skeletal muscle containing LDH5 have high demands for energy this means the large production of NADH and pyruvate from high substrate levels will not be metabolised by pyruvate dehydrogenase. The LDH5 (M form) is more specialised to convert pyruvate to lactate and ideal for anaerobic activities. Whereas the LDH1 form is more favourable for the conversion of lactate to pyruvate, LDH1 is predominantly found in the heart where there is an aerobic environment. This means despite increasing the substrate concentration, the LDH5 may not become as efficient in converting lactate to pyruvate as LDH1. The results reflect this as LDH1 has a generally greater enzyme activity with an increasing substrate solution compared to LDH5 (see table of results). In conclusion, the results obtained from the analysis of data on excel may not be highly accurate since most of the values have been rounded and it does not take into account any possible practical or human errors. Based on the charge of the two isoenzymes, a gel electrophoresis could have been done to calculate Vmax and Km. Bibliography: Goldberg E. (1972) Amino acid composition and properties of crystalline lactate dehydrogenase X from mouse testes. J. Biol. Chem. 247(7) pp 2046. Zhadin N, Gulotta M and Callender R. (2008) Probing the Role of Dynamics in Hydride Transfer Catalyzed by Lactate Dehydrogenase. Biophysical Journal. 95(4), pp 1975.

Bathing a Baby

———————– Bathing a baby 0-1 years old- At this very young age it is important that your baby is cleaned every night. This doesn’t mean that you should give your baby a bath every night, but you should at least ‘Top and Tail’ your baby. Topping and Tailing ‘Topping and Tailing’ means washing your baby from head to toe with warm, damp cotton wool. This method of washing doesn’t require a bath being made for the baby, however there are a few piece of equipment you will need to top and tail your baby. You will need to collect: †¢ Cotton wool pieces †¢ A towel A bowl with warm water (38 °c) †¢ A clean nappy †¢ Changing mat for nappy change How to Top and Tail your baby: 1. Gather all the equipment together before you begin. 2. Undress your baby down to their nappy and wrap them in a towel. Making sure their face isn’t covered. 3. The next step is to get a piece of c otton wool, dip it into the water and then clean your baby’s eyes, wiping from the inner corner outwards. It is extremely important to use a fresh piece of cotton wool for each eye, in case your baby has an infection which could be transferred from one eye to the other. . Next clean around their nose, the skin creases around his neck and under his chin, using a different piece of cotton wool. Then clean the contours of their ears, then their face. 5. You should then use the towel to dry the baby lightly. 6. You can then clean the rest if their body if you want, using a different piece of cotton wool each time. You can clean their hands, under their arms and around their belly button, and then you can remove their nappy. 7. Changing cotton wool frequently, you then need to clean the nappy area.You must always ensure that you wipe your from front to back on a girl to avoid transferring any germs to her genitals. 8. Lastly, you should pat your baby dry then put on a clean nappy and put their clothes back on. Bathing you baby becomes part of their daily routine. When your baby is young you may find that you bath them during the day. However, it is always good to make bath time part of the night time routine before your baby goes to bed. It is very important to understand how to bath your baby correctly, so that your baby is safe.Bathing your baby When bathing your baby you need get all the equipment together beforehand, such as: †¢ Cotton wool pieces †¢ A towel †¢ A baby bath with warm water (38 °c) †¢ A clean nappy How to Bath your baby: 1. Firstly, you should wash your hands and get all the equipment together that you need for bath time. 2. You should then fill the bath with cold water first, and then add hot water to get the bath to the right temperature (38 °c). To test the temperature you should use your elbow and the water should feel lukewarm. 3.Bring your baby to the bath area and remove your baby’s clothes. Wrap your baby in a towel, keeping their head exposed so you can wash their face and hair before you put them in the bath. 4. Wash your baby’s eyes, ears, face and neck as you would for topping and tailing. 5. Hold your baby so that their head is over the bath water and wash their scalp with a wet, soapy flannel or just water. Rinse their hair carefully and dry it. 6. Now take off your baby's nappy. If there are faeces in the nappy,  clean your baby's genitals  and bottom before putting them in the bath. . Gradually slip your baby into the bath feet first, using one hand to support their neck and head. The water should be covering their shoulders so that they don't get cold. 8. Use the cotton wool pieces to clean your baby from top to bottom, front and back. 9. Rinse your baby thoroughly, and then lift them out of the bath. Lift them with one hand supporting their neck and head and your other hand under their bottom. 10. Wrap your baby in a towel and dry them thoroughly, checking t hat folds of skin around the groin and neck are dry.Then put a clean nappy on them. You can then redress your baby. 1-3 years old- Toddlers tend to become messier and dirtier as they are now mobile. Therefore, it is really important that they are regularly having baths to keep them clean. Toddlers should be bathed every night and their hair should be washed at least once a week. When bathing your toddler it is very important that the water is not too hot or cold as this will make it uncomfortable for them. Therefore, you should check the water temperature using your elbow.You should also use a non-slip mat in the bath now so that the child does not slip and you should ensure that your toddler is not stand up in the bath It is essential that your child is never left unattended in a bath, as a child can drown very quickly in only a small amount of water. To wash your child you should use a child friendly liquid soap or body wash squeezed onto a wet sponge or cloth. You should use this to wash their body and then rinse off the excess soap. You should use a cup or a little bowl to rinse the soap off. You should always your child’s face, hands, and bottom.Cleaning their face and hands helps to get rid of germs and dirt. Washing your child’s bottom reduces the chances of nappy rash or irritation of the skin. You should save washing your child’s bottom till last and once you used the sponge on their bottom or other privates, set it aside and don’t use it again until you wash it. To wash your toddler’s hair you should use a children’s shampoo that won’t sting their eyes. To wash your child’s hair you should start by tilting their head back and pouring water over their head to wet the hair.You should then add a small amount of shampoo and should work it in the wet hair. Finally, you will need to tilt their head back, shield their eyes with your hand and rinse the shampoo out by pouring water over their head again. It is useful to have a dry towel handy to wipe away water and shampoo that gets in their face or eyes. Once your toddler has been washed you should take them out of the bath and you should dry them off with a towel. At this age it is important to let your child to start learning how to wash on their own.Therefore, you should talk through the routine with your child, telling them what you are doing at each stage. You could also let your wash themselves with the sponge and rub the shampoo into their hair; however you should always be close at hand in case they get soap in their eyes. You should also make bath time fun for your toddler by using lots of bubbles and bath toys, e. g. boats, rubber ducks, cups etc. Also, toys that squirt water are always fun for your child to play with, although the chances are that you may get a little wet! -5 years old- Your child should now be becoming more independent at bath time and may undress themselves for bath time. They will become more involve d in washing themselves, although you are still there to supervise them during bath time. They will also be familiar with the routine of bath time, although they may still make a fuss about bath time. Therefore, it is important that you make it as fun as possible for them. There are lots of different, new and exciting bath time toys for children to play with. You can buy soap crayons hich allow children to draw on themself, on the bath or on tiles. These crayons come in different colours and they wash off. You could also get Stick-on shapes to stick to the sides of the bath and walls. Also, bath time books are good. These books are waterproof and you can get involved in your child’s bath time by ready them the story. You can also make up games for them to play during bath time, like I spy, or you could make a treasure hunt by throwing lots of different toys into the bath for your child to find. -8 years old- Children around 6-7 years old are now capable of bathing or showerin g alone and should be given more privacy. You should teach your child how to turn on and off the taps and you should show them how to heat up the bath properly by turning on the cold water first, then gradually warm it up and testing it. You should also go over the basic safety rules of bath time, for example, don't hop in and out of the bath, use just a small bit of shampoo, and be extra careful getting in and out of the bath.You can now also help to teach your child on how to use a shower. It is important that you stay close and within earshot of the bathroom, in case of any problems but as long as your child knows how and what to clean, you should let them have some privacy. Your child is now in control of their own cleanliness, so you should ensure that your child knows how to fully rinse their hair, and you should keep on reminding them about washing everywhere, including behind the ears and down at the feet.

Notes on What Essay Topics Are on the Ged in Step by Step Order

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Alexander Hamilton And Thomas Jefferson - 819 Words

Alexander Hamilton was born on January 11, 1755 in Nevis, British West Indies and Thomas Jefferson was born on April 13, 1743, Shadwell, VA. They both grew up on plantations and had families and siblings. However, Jefferson had nine siblings while Hamilton only had his younger brother James A. Hamilton. The two men had a great adoration for reading and were brilliant. They were phenomenal writers and had many famous works. They were both founding fathers and were part of Washington’s first cabinet. Even though the two had quite a few similarities, the two could not be more contrasting. Once in Washington’s cabinet, the tension began and only escalated as time went on. Alexander Hamilton and Thomas Jefferson were destined enemies because they originated from differing backgrounds, economic ideals, and politics, which played a large role in them not being able to get along. 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