LAB 1 Flashcards
Mol to mmol
X1000
Mmol to mol
÷ 1000
mol to µmol
x1000000
µmol to mol
÷ 1000000
mmol to µmol
X1000
µmol to mmol
÷ 1000
L to ml
X1000
mL to L
÷ 1000
ml to µL
X1000
µL to ml
÷ 1000
µL to L
÷ 1000000
L to µL
X1000000
100 ÷ 1000 pipette
P1000 pipette, the first/top digit is thousands of µL, the middle digit is hundred, while the third is tens
1000 µL would read 100
350 µL would read 035
100-1000 µL
20 ÷ 200 pipette
P200 pipette, the first/top digit is hundreds of µL, the middle digit is tens, while the third is single units.
200 µL would read 200
95 µL would read as 095
20-200 µL
2 ÷ 20 pipette
P20 pipette, the first/top digit is tens of µL, the middle digit is single units, while the third is tenths.
20 µL would read 200
2.5 µLwould read 025
2-20 µL
Denaturation because of pH
At low pH ionisable side chains on amino acids become protonated, while high pH they become deprotonated. pH induced alteration of the ionisation status of a protein will result in the disruption of non covalent bonds that would normally stabilise the protein fold
Denaturation because of heat
Heating a protein will increase the kinetic energy of the molecules in the solution. When you add sufficient heat energy to the molecules they start to vibrate so rapidly that the weaker non covalent chemical bonds will be disrupted . The breaking of the non-covalent bonds in a protein can destabilise the structure and cause the protein to denature
Starch
Starch is a carbohydrate found in plants. It consists of two different types of polysaccharides that are made up of glucose monomer units which form two different structural arrangements. One is the linear but coiled amylose and the other is branched amylopectin.
Salivary amylase protein structure and function
Salivary amylase is an enzyme that initiates carbohydrate breakdown in the mouth, digesting starch specifically, hydrolysing polysaccharides into oligo-, tri- and disaccharides. As the partially digested starch reaches the stomach, the acidic conditions inactivates the salivary amylase, and pancreatic amylase is secreted into the small intestine subsequently hydrolyses the partially digested starch into disaccharides
Salivary amylase is a monomeric protein whose single polypeptide chain comprises of 496 residues. Its secondary structure includes a total of 20 alpha helices and 42 beta strands. These form functional domains including a beta barrel made up of 8 antiparallel beta strands surrounded by 8 alpha helices i.e. an alpha/beta barrel - this makes up the catalytic domain and a greek key beta barrel. In addition to non-covalent bonds, the territory structure of the enzyme is stabilised by 5 disulphide bonds.
Salivary amylase relies on several key residues to perform catalysis, including active site residues that act as a nucleophile and proton donor. There are also several histidine (His) residues that are essential in the recognition and binding of the starch substrate, as well as being involved in catalysis. Charged and/or polar amino acids such as arginine (Arg), asparagine (Asn), aspartate (Asp) and His are critical in holding the catalytically important Cl- and Ca+ ions in place
Iodine test
In a solution containing starch, iodine will bind to the amylose component of the carbohydrate, causing a colour change to dark blue/black. In the absence of starch, the iodine has nowhere to bind, so the colour of the solution will resemble to pale yellow/orange colour of the iodine itself.
Results of amylase experiment
Colour change in positive control (no amylase), 80 degree treated salivary amylase, HCl toasted salivary amylase (the last two are a result of the denaturing of the amylase)
Following heating of the amylase protein to 80C, we see a deep blue/black colour following the iodine starch test. This indicates that starch is still present in the solution and has therefore not been broken down by the amylase protein, suggesting that the amylase protein has been denatured by the high heat. We do not see this effect at 40C or room temperature. With regards to HCl treatment, we also see a colour change to deep blue/black, again indicating that acidic conditions caused the amylase protein to denature and therefore unable to function in its role of carbohydrate digestion.
What effect does heat have on chemical bonds?
High heat breaks the non-covalent bonds in the amylase causing it to denature
What happens when it is heated to 80 degrees?
At this temperature, a significant number of the non covalent bonds break which impacts on the function of amylase as it will be denatured and the hydrophobic core will now be exposed
The secondary and tertiary structure will now be affected
At low pH, explain how extensive protonation of ionisable side chains of salivary amylase affects the function of the protein
Low pH means lots of H+ - affects non covalent bonds - can cause structural change therefore change in function as well as exposing the hydrophobic core of the protein
Dilution factor
Concentration of undiluted solution A/ concentration of dilute solution A = whole number
Concentration of diluted solution A/ concentration of undiluted solution A = fraction
Dilution factor = total volume / volume of solution A
Dilution factor of 5 is also the same as …
1 in 5 dilution
1/5 dilution
5 fold dilution
1:4 dilution (1 part solution A to 4 parts solution B)
Solving dilution calculations using a formula
Initial concentration x initial volum = final concentration x final volume
Final = from the diluted solution
