Buffer
What is a buffer?
A solution that resists changes in pH when a little acid or base is added.
What is Beer's law (Beer-Lambert law)?
A = ε l c is known as this law.
These two aromatic amino acids contribute most to a protein's absorbance at 280 nm.
What are tyrosine and tryptophan?
The molecule an enzyme acts upon.
What is the substrate?
This inhibitor competes with the substrate for the active site. This is how it changes Km and Vmax.
What is a competitive inhibitor? Km increases and Vmax is unchanged.
The two solutions are acetic acid (pKa 4.76) and HEPES (pKa 7.5). This is the solution you would choose to buffer at pH 6.6 and why
What is HEPES because its buffering range is from 6.5-8.5
The unit of absorbance.
None. (Absorbance is unitless)
In the biuret assay, Cu2+ reacts with this bond in proteins in basic solution to give a complex of this color, measured at this wavelength.
What is the peptide bond, purple, 540 nm?
Besides substrate concentration, these two physical conditions affect enzyme activity.
What are temperature and pH?
This inhibitor binds somewhere other than the active site, doing this to Vmax, and leaves Km unchanged.
What is a noncompetitive inhibitor? what is lowering Vmax?
In a titration, this is the point where enough base has been added to completely neutralize the acid.
What is the equivalence point?
This curve is made from samples of known concentration and is used to find the concentration of an unknown from its absorbance. If the absorbance plateaus at high concentration, you do this with those points.
What is a standard curve? Leave out the points beyond the linear range.
Classify each as a direct or an indirect measurement: A280 of a protein; the biuret assay of a protein; the glucose oxidase/peroxidase assay of glucose. Then state the rule.
What is: A280 is direct; biuret is indirect; the glucose assay is indirect. Direct = you measure the absorbance of the substance itself. Indirect = you measure a colored product of a reaction (the Cu2+-peptide complex, or oxidized o-dianisidine at 440 nm).
Km is the substrate concentration at which the reaction velocity equals this.
What is half of Vmax (0.5 Vmax)?
This inhibitor binds only the enzyme-substrate complex. This is what happens to Vmax and Km.
What is both decrease (uncompetitive inhibition)?
A 10 mL sample of 0.05 M weak acid needs 15 mL of 0.1 M NaOH to reach its final equivalence point. This is the number of equivalents and the number of protons each molecule can donate.
What is 3 equivalents, so 3 protons? (5.0 x 10-4 mol acid; 0.1 M x 0.015 L = 1.5 x 10-3 mol NaOH; 1.5 x 10-3 / 5.0 x 10-4 = 3.0.)
A dye solution has A = 0.54 in a 1.0 cm cuvette. Its extinction coefficient at that wavelength is 6,000 M-1 cm-1. This is its concentration in µM.
What is 90 µM? (c = A / (ε l) = 0.54 / (6,000 x 1.0) = 9.0 x 10-5 M.)
Name one thing A280 and the biuret assay have in common, and two ways they differ.
What is: both measure absorbance with a spectrophotometer to find protein concentration (so both rely on the absorbance-concentration relationship of Beer's law)? Differences, any two: 280 nm (UV) vs. 540 nm (visible); direct vs. indirect; biuret needs reagent and a standard curve, the Nanodrop does not; 2 µL (0.5 mm path) vs. 0.5 mL (1 cm path).
Enzyme X reaches half of its Vmax at 20 µM substrate. Enzyme Y needs 200 µM. This enzyme grabs substrate more easily, and this is why.
What is enzyme X, because it has the lower Km (20 µM vs. 200 µM)?
A Lineweaver-Burk line is 1/v = 40 x (1/[S]) + 0.5, with 1/[S] in µM-1 and 1/v in (µmol/min)-1. These are Vmax and Km.
What is Vmax = 1 / 0.5 = 2.0 µmol/min and Km = slope x Vmax = 40 x 2.0 = 80 µM? (The x-intercept, -1/Km = -0.0125 µM-1, gives the same Km.)
A buffer has a pKa of 6.8. This is the ratio [A-]:[HA] at pH 6.8 and at pH 7.8, and this is the pH where capacity is highest.
What is 1:1 at pH 6.8 (log 1 = 0, so pH = pKa, highest capacity) and 10:1 at pH 7.8 (pH = pKa + 1, the edge of the buffering range)?
A standard curve for the glucose assay is A = 0.80 x [glucose in mM] + 0.02. A soda diluted 1000-fold gives A440 = 0.22, which is inside the linear range. This is the glucose concentration of the original soda.
What is 250 mM? ([glucose] in the diluted sample = (0.22 - 0.02) / 0.80 = 0.25 mM; 0.25 mM x 1000 = 250 mM.)
Your sample also contains DNA. This method may give a wrong answer, and this is why.
What is A280, because nucleic acids also absorb UV light (the Nanodrop reads proteins and nucleic acids), so the reading is too high? Bonus: biuret is the one that needs a standard curve.
Enzyme X has Vmax = 1.0 µmol/min and Km = 20 µM. Enzyme Y has the same Vmax but Km = 200 µM. Both are tested at [S] = 20 µM. This is the velocity of X, and this enzyme grabs substrate more easily.
What is 0.5 µmol/min (at [S] = Km, v = half of Vmax), and enzyme X, because it has the lower Km? For comparison, Y runs at 1.0 x 20 / (200 + 20) = 0.09 µmol/min.
In Figure C, find Vmax and Km without inhibitor. Then name the inhibitor type and say where the two lines meet.
What is: no inhibitor, y-intercept 1.0 so Vmax = 1.0 µmol/min, x-intercept -0.005 µM-1 so Km = 1/0.005 = 200 µM. With inhibitor, y-intercept 2.0 so Vmax = 0.5 µmol/min, same x-intercept so Km is still 200 µM. Vmax down, Km unchanged: noncompetitive. The lines meet on the x-axis at -1/Km.