Materials and methods

4.2.1 Materials

4.2.1.1 Chemicals

Water was deionized and processed through a Millipore MilliQ Plus system such that the resistance of the water was greater than 18.2 ohms. Trizma (Tris[hydroxymethyl]aminomethane), bovine serum albumin (BSA), porcine pancreatic elastase, and substrate (N-(methoxysuccinyl)-ala-ala-pro-val 4-nitroanilide) were obtained from Sigma (St. Louis, MO). The Bradford dye reagent was obtained from Bio-Rad Laboratories (Hercules, CA). All reagents were of the highest purity. The cotton used was pre-ground to 80-mesh size using a Wiley Mill. Fiber fragments from both samples were isolated using an 80-mesh screen as described [21]. Cotton derivatives consisted of carboxymethylated cellulose (CMC) and an underivatized sample. The cotton samples were prepared as described previously [5, 22]. The CMC derivative was prepared as described [20].

4.2.1.2 Equipment

HPLC: Quantitation was performed using high performance liquid chromatography (HPLC) with a BioRad Series 500 HRLC and a Bio-Rad column heater (Bio-Rad Laboratories, Hercules, CA). The detector was a Gilson 118 UV/Vis unit that was set to 280 nm for the detection of albumin (or elas-tase). The flow rate was 1.0 mL/min. The eluents used are shown in Table 4.1. Calibrations were carried out using a low-dead volume (LDV) connector in place of a column. An LDV connector is a fitting with a small flow volume that can be connected in place of any HPLC column. The only proteins studied were BSA and elastase. Calibration curves were generated for each protein.

Spectrophotometer: A Cole Parmer 1200 spectrophotometer was used for enzyme activity measurements. The wavelength was kept constant at 410 nm for enzyme kinetic measurements. Proteins were assayed using HPLC as described.

Optical Micrograph: Samples were placed on a glass slide then viewed using reflected light followed with the placement of a glass cover slip over the fibers to attempt to bring them into the same focal plane. The samples

Table 4.1. Peak areas of BSA in the HPLC system for each individual buffer system

Buffer conditions effect on BSA

Buffer

Tris

NaCl

pH

Peak area*

1

5mM

0

9.0

1.66 ± 0.039

2

5mM

0

7.4

1.66 ± 0.035

3

5mM

0.15 M

9.0

1.62 ± 0.079

4

5mM

0.15 M

7.4

1.59 ± 0.077

* Areas given are x 106 area counts/second. The difference in peak areas between buffer 1 and 4 is less than 5%.

* Areas given are x 106 area counts/second. The difference in peak areas between buffer 1 and 4 is less than 5%.

were then examined on an Olympus BX 60 light microscope (Olympus model BX60) at different magnifications and digital light micrographs were taken of the sample using a Kodak DC290 digital camera.

Scanning Electron Microscopy (SEM): Both cotton fiber samples were prepared for viewing in a field emission scanning electron microscope in the same manner. A 13 mm SEM stub was cleaned and a small piece of graphite tape was placed on the top of the stub. Then a small amount of each cotton fiber sample was adhered to the graphite tape. The material that had not adhered was removed with a small discharge of air from a filtered air source. The sample was then allowed to sit in a vacuum of the Edwards S150B Sputter Coater for 1 hour to allow for the small particles to settle on the graphite tape. After the particles had settled, the samples were sputter coated with carbon for 10 seconds. The samples were removed from the sputter coater and placed in a N2 fed dry box for storage.

The samples were viewed on the LEO 982 FESEM (Carl Zeiss, Inc., Thornwood, NY 10594) with varying magnifications. The LEO 982 FESEM has resolution capabilities of 1.0 nm at 30 kv. High and low magnification images were taken of the particles with the majority of the images being taken of common structures found in the majority of the samples. The samples were then removed from the FESEM and placed back in the dry box for storage and further examination.

4.2.2 Methods

4.2.2.1 Protein Quantitation

Four buffers were created in the pH range (7-9) of Trizma® salt to determine variability in absorbance readings over the operating range of the experiment. The buffer conditions can be seen in Table 4.1. Twenty microliters of 40 |xg/mL BSA in 5 mM Tris, 0.15 M NaCl pH 7.4 was injected with one of the four buffers in Table 4.1 eluting at 1.0 mL/min. The same method was used in all four buffer systems. Pumps were operated remotely through the ValueChrom® software and absorbance was measured at 280 nm as described above. Peak areas were calculated with the built-in integrator. Each buffer was eluted through two dual-piston pumps operating at 50% each.

The amount of protein adsorbed was determined by subtracting the peak area of the protein (BSA) or enzyme activity (elastase) that eluded through the cotton column from that eluted through the LDV connector. Elastase activities were determined by adding the activity of fractions collected downstream from the detector. Three 0.5 mL eluted fractions were collected, analyzed for enzyme activity, and totals were added to obtain the eluted activity. The dynamic range of this method was verified by determining the linearity of elastase activities of diluted enzyme.

Albumin was examined through subsequent injections of BSA in 0.15M NaCl, 5 mM Tris, pH 7.4 until the absorbance values were measurable and had stabilized to a constant value. Elastase was injected in a similar manner, but only after the cotton column had been pre-conditioned by injecting repeated injections of BSA as described above. Several injections of elastase were also put through the LDV connector. In all cases, the amount of elastase injected was nearly identical to the amount eluted.

4.2.2.2 Elastase assay

Elastase activity was measured using the substrate (N-(methoxysuccinyl)-ala-ala-pro-val 4-nitroanilide) and measuring the rate of change in absorbance at 410 nm. This method has been described previously [21]. A 600 micromolar solution of substrate was solubilized in dimethylsulfoxide (DMSO) and 100 microliters of this substrate solution was added to initiate the enzyme reaction. Assays were conducted in pH 7.4 buffer containing 5 mM Tris, 0.15 M NaCl, and the release of p-nitroaniline was measured at 410 nm from the enzymatic hydrolysis of the substrate. The initial slope of the increasing absorbance at 410 nm was used to determine K values.

The procedures used to determine the activity of elastase in the presence of albumin that had been bound to CMC and underivatized cotton samples were identical. A 570 |xg/mL elastase in 0.2 M NaCl solution was used. Elastase samples were injected into HPLC columns that had been conditioned with BSA as described above. The activity of each eluted fraction resulting from a single injection was determined and if several adjacent samples were active, the totals were added. Usually, only three consecutive 0.5 mL fractions contained measurable enzyme activity.

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