By C. Dennison

It is a truism of technological know-how that the extra primary the topic, the extra universally appropriate it's. Neverthelens, you will need to strike a degree of "fundamentalness" acceptable to the duty in hand. for instance, an in-depth examine of the mechanics of motor automobiles could inform one not anything concerning the dynamics of site visitors. site visitors exists on a special "level" - it truly is based upon the lifestyles of motorcars however the physics and arithmetic of site visitors might be safely addressed by way of contemplating motorized vehicles as cellular "blobs", for granted of ways they develop into cellular. to begin a discourse on site visitors with a attention of the mechanics of motorized vehicles may therefore be inappropropriate. In penning this quantity, i've got wrestled with the query of the fitting point at which to deal with the physics underlying the various strategies utilized in protein isolation. i've got attempted to strike a degree as will be utilized by a mechanic (with might be a moderate leaning in the direction of an engineer) - i.e. a pragmatic point, providing acceptable perception yet with minimum arithmetic. a few humans curious about biochemical learn have a minimum grounding in chemistry and physics and so i've got attempted to maintain it so simple as possible.

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Knowledge of the Km is useful when devising an enzyme assay as it enables one to use a substrate concentration where Vo will not be too sensitive to small changes in [S] due to experimental error. A good ruleof-thumb is that [5] should be as high as possible, preferably at a level where the substrate dilution curve is asymptotic to Vmax. Often, however. [5] is constrained by cost or experimental practicability, and values of less than Km may have to be used. For example the proteinase cathepsin B is routinely assayed at [S] = 410 K m, using a fluorogenic substrate.

8 • In measuring the concentration of proteins by their UV -absorbance, remember that the extinction coefficient (or absorption coefficient) is given by the equation:- A where, amcl A = c = am = absorbance molar extinction coefficient molar concentration of protein in solution length of the light path through the solution (usually 1 cm). 2 Gs Gm = specific extinction coefficient. GsxMW. The biuret assay In alkaline solution, proteins reduce cupric (Cu2+) ions to cuprous (Cu1+) ions which react with peptide bonds to give a blue coloured complex.

5. CH 3 COOH is the acidic species in this buffer and CH3COO- is the conjugate base. It may be observed that a solution of acetic acid itself (CH,COOH) will have a pH less than the pKa of acetic acid. Conversely, a solution containing only sodium acetate will have a pH greater than the pKa of acetic acid. 1. CH3COOH < CIi:3 COO -Na + pKa NaOHadded • Figure 5. Schematic titration curve of a monoprotic acid, such as acetic acid. A triprotic acid, such as phosphoric acid will yield a titration curve having three inflection points (Figure 6), corresponding to the three pKa values of phosphoric acid.

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