Year : 1970 | Volume
: 18 | Issue : 3 | Page : 106--110
Electrophoretic analysis of lacrimal fluid proteins and demonstration of lysozyme in normal human tears
SRK Malik, AK Gupta, R Berry
Maulana Azad Medical College, New Delhi, India
Maulana Azad Medical College, New Delhi
|How to cite this article:|
Malik S, Gupta A K, Berry R. Electrophoretic analysis of lacrimal fluid proteins and demonstration of lysozyme in normal human tears.Indian J Ophthalmol 1970;18:106-110
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Malik S, Gupta A K, Berry R. Electrophoretic analysis of lacrimal fluid proteins and demonstration of lysozyme in normal human tears. Indian J Ophthalmol [serial online] 1970 [cited 2021 Jun 17 ];18:106-110
Available from: https://www.ijo.in/text.asp?1970/18/3/106/35073
The study of protein fractions of normal human tears was carried out at first by Smollens, Leopold and Parker (1949) using free electrophoretic technique of Tiselus.
Junnola (1952) using nephelometric method studied protein components of tears in thirty normal individuals and found values ranging from 136-592 mg/100 c.c. with an average 360 mg/100 c.c.
Fractionation of lacrimal fluid proteins utilising the technique of electrophoresis has been performed by many workesis. McEwen and Kimura (1955) and Erickson, Berg and Hatlen (1959) obtained three protein fractions while Brunish (1957) demonstrated five major fractions in normal human tears. Krause (1959), however, obtained six fractions by means of a paper electrophoresis.
The fractionation of lacrimal fluid proteins by agar gel electrophoresis has been studied only by Francois and Rabaey (1960) and Tapaszto and Vass (1965).
In our study, the technique of agar gel electrophoresis has been used to analyse lacrimal fluid proteins and demonstration of lysozyme.
Methods and Materials
The lacrimal fluid was collected by means of a fine pipette- causing minimum irritation to the eye. About 0.25-0.5 cc. of lacrimal fluid was collected and used for electrophoresis.
Preparation of Agar Gel:
4 gms. of agar (Difco) was added to 100 c.c. of distilled water taken in a water bath to dissolve the agar completely. The dissolved agar was spread on a glass plate and allowed to set. One centimeter wider strips of agar gel were cut and repeatedly washed with distilled water. 100 gms. of purified agar were dissolved in 166 c.c. of half diluted buffer (PH 8.2, Mo 0.05) by heating on a water bath. This was filtered through eight thickness gauze and used for subsequent experiments.
Preparation of Vernol Buffer:
33 grams of sodium barbitonc were dissolved in two litres of distilled water. 3.5 c.c. of 12 N.1-Icl. were added to it and the contents of the bottle were thoroughly shaken. The pH of the solution was adjusted to 8.2 by adding more Hcl if needed.
Preparation of Slides:
4-5 c.c. of molten agar was poured on the slide and the agar allowcd to set. A slot was made in the centre and the lacrimal fluid to be analysed was put into the slot with the help of tuberculin syringe.
The protein fractions were separated by agar gel electrophoresis using buffer of pH 8.2, Mo 0.05 at a potential difference of 30 Volts. on the ends of the slide (7.5 cms. by 2.5 cm.). The current was allowed to pass for 1.5 to 2 hours.
Fixation of Proteins:
The slide was taken out of the electrophoretic chamber and immersed in a fixation fluid (98 c.c. of rectified spirit and 2 c.c. of glacial acetic acid).
Drying and Staining of Slide:
The slide, after fixation, was dried at room temperature and stained with 1% amido black stain for 15 minutes and washed till the background was clear and then kept for drying.
Quantitative Estimation of Proteins:
The quantitative estimation of various protein fractions was determined with the help of Amil densitometer having a photosensitive cell. A graph was plotted and percentage of various fractions calculated.
Before starting, the electrophoresis of lacrimal fluid, the procedure was standardised on normal human serum. [Figure 1].
Demonstration of Lysozyrne:
In Order to demonstrate the lysozyme fraction the agar gel slides were taken before the fixation of proteins. The agar gel from anode and cathode ends were collected in two different test tubes. A culture of saprophytic staphylococci was taken and put in both the test tubes containing agar gel. This was incubated at 37°C for 2 hours and then cultured on blood agar plates. The blood agar plate was divided into four quadrants. In the first quadrant fraction from the anode end and in the second quadrant agar gel from the cathode side was inoculated. The third quadrant was inoculated with culture of saprophytic staphylococci and the fourth quadrant was left blank.
This plate was incubated at 37˚C for 24 hours and then examined. The fraction containing lysozyme will cause lysis of bacteria and hence that quadrant will show no growth while the quadrant devoid of lysozyme will have unrestricted growth of bacteria.
The electrophoretic pattern of lacrimal fluid proteins in normal human beings revealed five major fractions with a maximum of eight subfractions. Fractions I and II moved towards the cathode while the fractions III, IV and V and moved towards the anode [Table 1], [Figure 2].
On comparing the mobilities of normal lacrimal fluid proteins with that of normal human serum it was seen that fraction I had the mobility corresponding to gamma globulins, fraction II between alpha 2 and beta 1 globulins, fraction III had the same mobility as alpha 1 globulins, fraction IV had the mobility corresponding to serum albumin and fraction V had the mobility which was slightly higher than that of serum albumin.
On examining the four quadrants of the culture plate [Figure 3] after 24 hours (already described in methods and materials), it was observed that quadrant `A' which represented fraction from the anode side showed growth of saprophytic staphylococci: quadrant `S' in which normal saprophytic staphylococci were inoculated also showed positive growth; quadrant `C' which contained the fraction from the cathode end did not show any growth indicating that lysis of saprophytic staphylococci had occurred and the quadrant `H' which was left blank at the time of inoculation did not show any growth.
Hence it was observed that lysozyme was present in the: fraction immediately next to the slot i.e. fraction 11 which moved towards the cathode side.
The fractionation of lacrimal fluid proteins by agar gel electrophoresis has been studied by Francois and Rabaey (1960) and Tapaszto and Vass (1965). We also carried out the technique of agar gel electrophoresis because of following advantages (i) quantity of solution required is minimal and (ii) Protein fractionation is much more detailed than by other techniques.
In our series results of electrophoresis of normal lacrimal fluid revealed five major fractions with a maximum of eight subfractions. This observation agrees with the observations of Brunish (1957) who also obtained five major fractions by filter paper electrophoresis. Our observations also support the work of Tapaszto and Vass (1965) who demonstrated 8-10 subfractions in five major protein groups by means of agar gel electrophoresis. McEwen and Kimura (1955) and Erickson, Berg and Hatlen (1958) obtained six fractions in normal human tears by paper electrophoresis. Francois and Rabaey (1960) could demonstrate four major fractions by agar gel electrophoresis.
The electrophoretic analysis showed that normal tears had lysozyme component which moved towards the Cathode as confirmed by lytic activity and was found to be associated with fraction II. This finding agrees with the findings of many other workers who also demonstrated the mobility of lysozyme towards the cathode side. (Smollens, Leopold and Parker, 1949; Migliar and Pirroda 1954; McEwen and Kimura 1955; McEwen, Erickson et al 1959; Krause 1959 and Tapaszto and Vass 1965). Francois and Rabaey (1960), however, identified the lysozyme in the fraction moving towards the anode end. They postulated that lysozyme does not migrate normally in agar due to the interaction between agar and the enzyme but in our series lysozyme moved normally towards the cathode side perhaps because of temperature difference which was lesser i.e. 4°C and voltage difference i.e. 30 v/cm. instead of 10 v/cm. The duration of current was also more in our series which might have some influence on the mobility.
Summary and Conclusions
A study of electrophoretic analysis of normal human tears and demonstration of lysozyme was performed utilising the technique: of agar gel electrophoresis.
A total of five major fractions with eight subfractions could be demonstrated in normal human lacrimal fluid.
The mobilities of these fractions were studied and compared with those of serum.
Lysozyme was demonstrated by the lytic activity which was found to be associated with fraction II and moved towards the cathode.
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