Does John Have an Acid-Base Disorder? Explain

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Serine is classified as a polar amino acid with a hydrogen group and is small in size. The changing serine to another amino acid often has little effect because serine is quite common in protein functional centres. The hydroxyl group is fairly reactive and hence being able to form hydrogen bonds with a variety of polar substrates (Reva, 2011). Also, its small size means that it is relatively common within tight turns on the protein surface. As a result, serine can be substituted by other polar or small amino acids. For example, serine can be replaced to threonine since both of them are hydrophilic amino acids and are small in size. This will not alter the structure very much and the protein is still working (Reva, 2011). Both of them have same chemical properties and therefore have the same function and role. One of the common roles for serine and threonine is phosphorylation (Reva, 2011).

Q4.

These hormones may be extracted from bovine pituitary glands for therapeutic use. It is suspected that a commercial product contains a mixture of the two hormones, rather than a pure sample of oxytocin. Suggest appropriate tests that could be used to check product purity.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is the most widely used gel electrophoresis technique that used for proteins. SDS-PAGE is used to estimate and check the purity of a protein sample such as oxytocin (Shapiro, 1967). SDS-PAGE runs in the presence of negatively charge detergents (sodium dodecyl sulphate) that serve to disrupt most of the secondary, tertiary and quaternary structure of proteins. Because the charge-to-mass ratio is nearly the same among SDS-denatured proteins, the final separation of proteins to determine the purity is almost entirely dependent on the differences in relative molecular weight of oxytocin (Klaus & Mary, 1969).

SDS-PAGE consists of a piece of SDS-polyacrylamide gel for samples to be loaded into the well. An electric field is then applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel towards the positive electrode (the anode) because SDS is negatively charged (Klaus & Mary, 1969). The protein migrates only depending on its size (molecular weight), each biomolecule moves differently through the gel matrix. The smaller the size of a protein, the faster it moves towards the anode because small molecules more easily fit through the pores in the gel while a larger protein may encounter more resistance, hence move slower (Lutz, 1977). As a result, it can be said that SDS-PAGE is an effective way to screen for the purity of a protein.

Q5.

(a) Those with one disulphide bridge denature at temperature 2-7°C higher than the wild-type lysozyme, while those with two disulphide bridges denature at temperatures 7-10°C higher than the wild-type lysozyme.

This result showed that the lysozyme with two disulphide bridges is more heat stable than lysozyme with one disulphide bridges and without disulphide bridges. The inserted disulphide bonds had increased the thermostability of the lysozyme, meaning that the protein can withstand a higher temperature, hence has a higher resistance towards thermal denaturation. This is because the energy required to break the bond increase. The disulphide mutant can be said is fully active and retains its activity at substantially higher temperature than the wild type lysozyme (Signor et al., 1989).

(b) Another mutant form of lysozyme, in which proline was substituted for glycine, has a significantly increased Tm2.

Proline has very low melting point among amino acids as it destabilize the double helix. This is because proline cannot form a regular a-helix due to steric hindrance arising from its cyclic side chain. Also, the lack of a hydrogen in proline structure chemically prevents it from participating in hydrogen bonding. As a result, it is a less stable structure and side chain packing is less favourable (Chadalavada et al., 1997). However, glycine is a preference to substitute for proline because glycine was found to stabilize the double helix and hence increase the Tm. The helix is stabilized by allowing all the NH or CO groups within the polypeptide backbone to form hydrogen bonds. This bonding is considered energetically favourable hence more energy is needed to use to break the hydrogen bonds during high temperature (Schellman, 1987).

(c) Yet another mutant form of lysozyme had aspartate substituted for serine at the N-terminal of an α-helical segment. The Tm increased by 2°C.

Aspartate was substitute for serine in order to enhance the thermal stability of the enzyme. Calcium is essential for folding in many proteins. The substitution of aspartate for serine had increased the calcium-binding affinity of the amino acid and hence improved the thermostability, subsequently increased the Tm. The N-terminal of an α-helical segment may contribute to a stronger calcium binding site. Besides, more calcium ions contribute to the increase in overall stability of the surface region and enhance thermal stability of a protein (Thornton, 1981).

References

Berend, K., Aiko, P. J., Rijk, O. B. & Gans, M. D. (2014). Physiological approach to assessment of acid-base disturbances. The New England Journal of Medicine, 371 (2): 1434-1445

Chadalavada, S. V., Tangirala, S. & Arjula, R. R. (1997). DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process. Federation of European Biochemical Societies, 410 (3): 201-205

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Klaus, W. & Mary, O. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. The