Solidification/Stabilisation (S/S)

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a sheet of paper. The lens capsule was in turn removed by incising the lens at the equator and picking up the capsule with a pair of tweezers. The lenses were partially dissolved in 20ml phosphate buffer pH7.1 and shaken for 8 and 12 hours respectively depending on the age of the lenses using a mechanical shaker. The lenses of 4 goats with age of 2 years were shaken for 8 hrs, while 3 yrs old lens shaken for 12 hrs.

2.2.2 DETERMINATION OF α-CRYSTALLIN

The dissolved lenses were homogenised using Potter Elvejhem homogenizer, and centrifuged at 20000rpm for 20 minutes. The supernatant lens’ homogenate contained the water soluble proteins, including the alpha crystallin. The absorbance of the supernatant was measured at 280 nm and its concentration calculated using the formula: Conc. = Average absorbance / Extinction coefficient.

2.2.3 ESTIMATION OF PROTEIN BY FOLIN-CIOCALTEU [LOWRY]

METHOD (1951) Principle

The Folin-Ciocalteu method is a colorimetric method which is sensitive detecting down to 10 µg/ml of protein, and the sensitivity is moderately constant from one protein, to another. When the Folin reagent (a mixture of sodium tungstate, molybdate and phosphate), together with a copper sulphate solution, is mixed with a protein solution, a blue-purple colour is produced which is analyzed in the visible range of 600-660nm.

Procedure

Ten clean test tubes were serially labelled1-10.The first was used as blank no bovine serum albumin (BSA) added to it. From the second tube to the last 0.1ml up to 0.9ml of BSA and phosphate buffer was added respectively (Table 1), 5ml of alkaline copper

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reagent (4% copper sulphate in 4% sodium potassium tartarate and 4% carbonate) was added and tubes incubated at room temperature for 10min. Then, 1.0ml of the Folin Ciocalteu reagent was added and incubated for 10min. While the tubes were being incubated, 2.0ml of the alpha crystallin solution was pipetted and the absorbance of the reaction mixture was read at 280nm.

TABLE 1: Observation Table for BSA Standard Curve.

Test tube

BSA (ml)

Buffer (ml)

[BSA]

(mg/ml)

A CU Reagent

Incubate at room temperature for 10mins

Forlin reagent

Incubate at room temperature for 10 min.

Blank − 1 0.00 5 1

1 0.1 0.9 0.03 5 1

2 0.2 0.8 0.05 5 1

3 0.3 0.7 0.08 5 1

4 0.4 0.6 0.10 5 1

5 0.5 0.5 0.13 5 1

6 0.6 0.4 0.16 5 1

7 0.7 0.3 0.20 5 1

8 0.8 0.2 0.20 5 1

9 0.9 0.1 0.26 5 1

2.2.4 PURIFICATION OF CRYSTALLIN USING GEL CHROMATOGRAPHY Principle

Column chromatography separates mixture of compounds through partitioning between two immiscible phases; the mobile and stationary phase. The stationary phase is bound onto a support or matrix while the mobile phase runs along the stationary phase.

Column preparation

The column XK 16/70, was washed with chromium azide and dried in an oven for 5 minute. A glass wool was soaked for a day, cut accordingly to size and inserted into the column gently with a glass rod. Three glass beads were used to prevent bubbles. Over head tap was placed on top of the column. Buffer was used through a giving-set to

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flush/clean the column before administration of the gel. The buffer was run 3 times so that the wool settled without any space. The gel mixed with phosphate buffered-saline was applied into the column, with continuous pouring of the buffer on top of the gel.

The buffer was run through the column in 2 phases. In the first phase, it was run and collected at a flow rate of 1ml per minute for 24 hrs. In the second phase, it was run the next day at flow rate of 2.3ml per minute to allow the gel to polymerise.

Preparing the gel suspension

All materials were equilibrated to room temperature. Sephacryl high resolution medium was shaken and made even slurry. Fifteen millilitres (15ml) of the medium slurry were poured into a graduated cylinder, which was then diluted with phosphate buffer to 200/ml. The medium was stirred with a glass rod and the homogenous suspension was made free from aggregates.

Column assembly

The medium slurry was resuspended and poured into the column in one continuous motion. The slurry was poured down with a glass rod held against the wall of the column, which prevented the introduction of air bubbles. The reservoir top was screwed tightly and connected to the pump, column out let was then opened.

Column equilibration

The column was equilibrated with 2000ml buffer. The maximum flow rate of 2.3 ml/min was maintained during the equilibration.

Buffer

Buffers composed of 7.05g sodium dihydrogen phosphate (NaH2PO4) in 1 litre of distilled water; and 7.95g disodium hydrogen phosphate (Na2HPO4) in 1 litre of distilled

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water was used. The buffers were chosen to match the requirement of the sample. To ensure long column life all buffers were centrifuged and filtered.

Purification of alpha crystallin

Two millilitre (2ml) of lens homogenate was applied to the gel-filtration column that was suspended which contained matrix. The proteins were eluted with phosphate buffer.

Seventy five (75) fractions were collected and the absorbance of each fraction was measured at 260nm, two millilitres per minute for two hours.

2.2.5 TEMPERATURE DESTABILIZATION OF OXIDIZED LYSOZYME AND CHAPERONE EFFECT OF ALPHA CRYSTALLIN

Four solutions were prepared in 4ml quartz cuvettes.

Solution 1 contained 0.2mg/ml lysozyme in phosphate buffer.

Solution 2 contained 3-fold quantity of alpha crystallin (0.6mg/ml) in phosphate buffer.

Solution 3 contained 0.2mg/ml lysozyme in phosphate buffer and 30mM DTT.

Solution 4 contained 0.2mg/ml lysozyme, 0.6mg/ml alpha crystallin in phosphate buffer and 30 mM DTT.

The effect of temperature was visualized by means of measuring absorbance with a spectrophotometer (in the range 240-400nm) in step of 20nm. The temperature of the solution was changed in step of five degrees, from room temperature to forty five degrees (30, 35, 40 and 45˚С), by circulating water from water bath.

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CHAPTER THREE