The following sections give detailed outline of the assay techniques used: (all assays were performed in triplicates)
2.3.1 Protein Assay
The protein assay used was the Bio-Rad protein assay. The assay is based on the shift in absorbance from 465 to 595 nm which occurs when Coomassie Blue G-250 binds to proteins in an acidic solution. Bio-Rad Protein Assay is based on the Bradford method (Bradford, 1976). All protein readings were corrected for solids content (see section 2.3.3).
Standard Assav Procedure
Several dilutions of Bovine serum albumin (BSA) protein (fatty acid free, Sigma Chemicals Co, Poole, Dorset) standards (0.2 - 1.4 mg/ mL) were prepared. A standard curve was set up each time the assay was performed. The assay was performed at room temperature.
0.1 mL of protein standards and a "blank" (i.e. sample buffer) were placed in clean, dry disposable 4.5 mL cuvettes. 3 mL of dilute dye reagent was added to each cuvette (dye reagent concentrate was diluted five times that of original concentration). The contents were mixed well by gentle inversion of the cuvette avoiding excess foaming. After a period of 15-20 minutes the absorbance at 595 nm versus reagent blank was measured and the 595 nm absorbance reading versus concentration of the standards plotted. The unknown protein concentration of the samples was read from the standard curve. The highest level of variability with a standard deviation of 6% of the mean value was found.
2.3.2 Enzyme Assay
The assay was according to Bergmeyer et a l (1983). The alcohol dehydrogenase activity assay mix was prepared using the compounds listed in table 2.3 combination of ingredients made up to 500 mL using deionised water.
To determine ADH activity 3.0 mL of the assay mixture was taken and added to 0.05 mL of diluted homogenate supernatant solution. The reaction was immediately monitored at 340 nm using a spectrophotometer (Beckman DU 64, Beckman, High Wycombe, UK).
The highest level of variability with a standard deviation of 6% of the mean value was found.
Table 2.3 ADH assay compounds
Compound Amount Ethanol 17.5 mL Tris buffer 2818.00 mg NAD^ 597.06 mg Glutathione 153.65 mg Semicarbizide 345.65 mg
ADH assays were only used to ensure that the protein released during high pressure homogenisation was intracellular and not cell wall protein.
2.3.3 Solids Correction Factor
The solids content of the homogenate varied depending upon the degree of disruption the suspension had been subjected to. It was necessary to compensate for the change in solids content of the sample. The method used in this study was based on the procedure followed by Hetherington et al {\91\). In this study an empirical model was defined based on work by Hetherington et al {\91\) 'm order to simulate the value of F, the solids correction factor, for varying initial yeast concentrations, Cj.
From experimental data published in Hetherington et. al. (1971) it was observed that, F, the solids correction factor was a function of the initial yeast concentration ( C i ) and also the degree that homogenisation is used. Thus,
F = f(C i,N ,P ) (2.1)
where C, is the initial yeast concentration (g/L); N is the number of passes through the homogeniser and P is the operating pressure of the homogeniser. From Hetherington's 1st order protein release model, the above equation can be simplified to:
F = f ( C i,C ) (2.2)
Where Cs is the protein concentration of the supernatant (mg/mL). Hetherington (1971) plotted F against Cs for varying Q. Based on these results a plot of F against Q was obtained for varying Cs; a linear relationship between F and Q was observed. A linear model of the form:
F = -a*x+l (2.3)
was applied to these plots. The slopes, a, where then plotted against corresponding Cs values to give an exponential curve of the form:
a = 0.5(l+exp(-Cs/15) (2.4)
Hence, if relationship found in equation was substituted back into equation the final empirical model is shown in equation.
F = (-Ci/2(l+exp(-Cs/15))+l (2.5)
Ci for a particular set of experiments will be a constant. Using equation 2.5 it is now possible to simulate values for F give Cs It was found that there was on average a ±4% difference between the experimental and simulated data.
2.3.4 Electrical Sensing Zone Measurement (ESZ)
Whole yeast cells and homogenate particle size distributions were measured using the Electrical Sensing Zone (ESZ). In the electrical sensing zone measurement (ESZ), particles suspended in an electrolyte are caused to flow through a small orifice in a non-conductive material, along with an electric current. Particles traverse the orifice essentially singly, causing electrical pulses at rates fi’om a few thousand to a few hundred per second or lower, depending upon the flow velocity, orifice size and particle concentration. The amplitude of each pulse is directly proportional to the volume of the particle as sensed by its "electrical envelope" displacement within the sensing orifice. When diameter is the expressed dimension for ESZ size, it therefore compounds to an diameter of the equivalent sphere.
Equipment Description
The instrument was an ELZONE model 280 PC (Particle Data Ltd., Cheltenham, Gloucs., U.K.) linked a data acquisition system. Figure 2.4 shows the layout of the computerised ELZONE system.
The analyser was fitted with a 30 um orifice tube and calibrated using latex standards of sizes 2.02 mm, 5.00 mm and 10.18 mm to give a 128 size ranges between 1-17 mm. The ESZ measurement technique used was not sensitive below 1.0 mm. The size range obtained is discussed in more detail in section 3.1. A smaller 18 mm orifice tube was initially used to give smaller channel sizes, however, blockage became a time consuming problem.
The filtered electrolyte used for suspending and diluting the particles was 0. IM KH2PO4
and 0.15M NaCl, at pH 6.5. The electrolyte was filtered twice and then degassed, using 0.1 mm cellulose nitrate filters (Whatman, Maidstone, U.K.) and a vacuum filter apparatus (Millipore, Watford, U.K.). Coincidence counts were reduced to <1% by using adequate dilutions of particle sample. All PSDs were corrected for electrolyte background noise. The PSDs were also corrected for any particles that had not originated from the whole yeast cells. In all the experiments three replicate counts were made to ensure consistency of results. The PSD data were stored as ASCII files and imported into EXCEL v3.1 for
fiirther statistical analysis. The highest level of variability with a standard deviation of 4% of the mean value was found.
2.3.5 Computational Analysis
All computational work was carried out on an IBM PS/2 Model 70 (IBM, Basingstoke, UK). Simulation and modelling work was validated using the MicroSoft EXCEL version 5.0 (Microsoft Corporation, One Microsoft Way, Redmond, WA 98052-6399), MicroCal Origin V 3.5 (Northampton, MA 01060 USA) and BBN RSl/RS Explore (BBN, London, UK).