Practical Enzyme Kinetics A Biochemical Laboratory Experiment H. Alan Rowe and Morris Brown Norfolk State University, Norfolk, VA 23504 Enzyme kinetics is a subject often omitted in many high school and undergraduate chemistry laboratories due to the lack of equipment andlor the complexity of demonstrating the subject. Many enzymes and substrates are expensive and short-lived and require specialized assays and equipment to maintain and monitor the enzvmatic reaction. This paper . . describesan experiment that pro\,idesa fundamental understanding of the kinetics of the enzvme ~ a n a i n The . experiment oitlined can he conducted uiing readily available, inexpensive materials and with a minimum of equipment. Papain (3.4.22.2) is a sulfhydryl protease that is isolated from the fruit of Carica papaya, the papaya? I t has been used as a meat tenderizer and more recently as an agent for the cleansing of "soft" contact lens. This enzyme is supplied in tablet form and reconstituted in distilled water to remove protein deposits from the surface of contact lens.2Azocoll is used as the substrate for papain in this experiment. Azocoll is insoluble, powdered cowhide to which a red dye has been covalently attached. When the peptide linkages of the collagen of the cowhide are hydrolyzed, the azo dye is released into solution. The rate a t which the dve is released is measured spectropbotometrically at the dye ahsorbance maximum of5?0 nm. Azocoll has been used to detect nonsprcific proteolytic activity in a variety of proteases such as; collagenase, pronase, trypsin, etc.3 One enzyme tablet (Allergan Soflens) was dissolved in 10 mL of double distilled water at room temperature (protein concentration approximately 0.5 mg/mL'). Azoco11(12.5 mg) was added to a spectrophotometer cuvette, and the reaction was started by the addition of a 2-mL aliquot of the enzyme solution to the cuvette (t = 0). The cuvette was inverted and the absorbance and time measured 30 s later. After each absorhance measurement, the cuvette was inverted and 30 s allowed to pass before the subseauent measurement. The 30-s waitine period was to allow ihe undigested, insoluble, collagen-azo&e particles to settle in order to obtain a stahle ahsorhance readin'. This procedure was repeated until four successive, identical absorbance measurements were obtained. The same procedure was followed for 7.1,4.1,3.4, and 0.98 mg of Azocoll. Figure 1shows the increase in absorbance a t 520 nm with time for each of the five substrate concentrations. To determine the initial velocity of each of the reactions, the initial slope of each curve was determined. The slope was calculated for the linear part of each curve between t = 0 and t = 8.0 min. In order to assess the kinetics of the enzyme papain with Azocoll as the substrate the data was analyzed using a Lineweaver-Burk plot (Fig. 2). The parameters of V,., and K, for the reaction were determined using this graph. This enzyme reaction seemed to follow simple Michaelis-Menten kinetics. The maximum velocity, V,,,, was 0.26 As2o/min, Preliminary data from this paper were presented at the Virginia Academy of Science meeting in Norfolk, VA (May 1987). Decker, L. A,. Ed. Worthington Enzymes: Freehold: New Jersey.
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Korb, D. R.. Greiner. J. V., Finnemore, V. M., Allansmith. A. R. Arch. Ophthalmol. 1983, 101. 48-50. Calbiochem Catalog 1985. 314-315. Westerik, J. 0.;Wolfenden, R. J. Biol. Chem. 1972, 247, 8195.
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Figure 1. Olaph of Am, versus time for the reaction of papain and Azocoll. A = 0.98. 0 = 3.4. C = 4.1. D = 7.1. and E = 12.5 rng of Arocoll. Papain concentration was constant for each reaction.
Figure 2. Lineweaver-Burk plot, l/vo (min X AAsg0-') versus l / S ( L . g-'). and K, equaled 1.56 g/L. The V,, and K , were determined from Figure 2 as the reciprocal of the y intercept and the negative reciprocal of the x intercept, respectively. One data point on the Lineweaver-Burk plot was found t o lie off the line. This may be due to the inaccuracy of weighing of 0.98 mg of substrate or the difficulty in obtaining a meaningful measurement of the initial velocity from Figure 1. This laboratory experiment can he accomplished simply and accurately using only very basic laboratory supplies and equipment. I t can he conducted as a laboratory or lecture demonstration by exhibiting the deepening of the red color
with time. Many variations of this experiment can also be performed: a set of cuvettes with a constant amount of an inhibitor of papain can be included to demonstrate competitive or noncompetitive inhibition using the LineweaverBurk plot, the enzyme can be reconstituted in a salt solution instead of double-distilled water, the pH and/or temperature may be changed to demonstrate the effects of these parameters on enzyme activity, cysteine or other sulfhydrylSluyterrnan, L. Biochim. Biophys. Acta 1967, 137. 430
containing molecules may be added to activate papain, and other proteolytic enzymes (chymopapain, trypsin, chymotrypsin, pepsin, pronase, etc.) can also be examined kinetically in this manner.5 Analogous experiments can be performed with other commercially available enzymatic contact lens cleaning preparations such as those which contain pancreatin or subtilisn. Acknowledgment
The authors wish to thank Michelle Tate for her assistance on this project.
Volume 65
Number 6
June 1908
549
