Purification of staphylocidal beta-lysin from rabbit serum

Our plain-English summary of Purification of staphylocidal beta-lysin from rabbit serum, by F.B. Johnson, D.M. Donaldson (Journal of Bacteriology, 1968).

The study

• Authors: F.B. Johnson, D.M. Donaldson
• Journal: Journal of Bacteriology
• Published: 1968;96(3):589-95
• Cited by: 31 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

A cationic protein of rabbit serum bactericidal for Staphylococcus aureus was purified. The specific activity per unit of protein of the purified staphylocidal preparation was approximately 37,000 times greater than that of the serum from which it was isolated. Similar techniques were used to purify serum beta-lysin active against Bacillus subtilis approximately 24,000 times. The staphylocidal activity cannot be attributed to the same beta-lysin active against B. subtilis, lysozyme, or antibody-complement systems. The concentrations of staphylocidal beta-lysin in the sera of the five mammalian species studied did not correlate with their beta-lysin activities against B. subtilis. The two beta-lysins are similar in that both were heat-stable, sensitive to trypsin digestion, had molecular weights near 6,000, and were found in higher concentrations in serum than in plasma. Furthermore, similar techniques can be used to absorb and elute both substances in highly purified forms using cellulose asbestos filter pads and ion exchange chromatography on carboxymethyl cellulose. In contrast to the beta-lysin against B. subtilis, the staphylocidal beta-lysin was not released from blood platelets, and it was inactive in the presence of heparin, sodium citrate, sodium oxalate, ethylenediaminetetraacetic acid, acidic phospholipids, and acid pH values. A variety of proteins, including those of normal serum, preferentially inhibited the bactericidal activity of staphylocidal beta-lysin but not the beta-lysin against B. subtilis.

In plain English

This 1968 laboratory study does not study asbestos exposure or disease; asbestos appears only as cellulose asbestos filter pads used to purify serum proteins. The authors purified two germ-killing beta-lysins from rabbit serum, one acting on Staphylococcus aureus and one on Bacillus subtilis. Survival of 24 coagulase-positive S. aureus strains in undiluted rabbit serum ranged from 0.14 to 68%; the purified substance killed all strains better. Of five species' sera, only horse and rabbit had measurable staphylocidal beta-lysin in the standard test, where one unit kills 99% of the bacteria. Specific activity (units per milligram of protein) rose to 37,000 times that of serum for the staphylocidal substance and 24,200 times for the B. subtilis substance, a gain per unit of protein, not in total activity. The authors noted that the first figure also reflects removal of inhibiting serum substances. Both substances survived heating and had molecular weights of 5,500 to 6,000. Only the staphylocidal substance was blocked by heparin, citrate, oxalate, EDTA and proteins. The authors concluded that it is a distinct bactericidal agent. They noted that their preparations still contained both substances.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) purification and characterization experiment on blood serum proteins
• Who was studied: Twenty-four strains of coagulase-positive Staphylococcus aureus (hospital isolates and phage-typed strains), Bacillus subtilis, and sera from five mammal species (rabbit, guinea pig, human, horse, rat). The work was done at Brigham Young University, Provo, Utah, and published in 1968.
• Exposure: No asbestos exposure was studied. Asbestos appears only as cellulose asbestos filter pads, which were used as a laboratory tool to capture and purify the serum beta-lysins.
• Main results:
  • Across the 24 S. aureus strains, 2-hour survival in undiluted rabbit serum ranged from 0.14 to 68%.
  • Strains that were quite resistant to normal serum were highly susceptible to the purified beta-lysin.
  • Of the five species' sera tested, only horse and rabbit sera had measurable staphylocidal beta-lysin in the standard assay, in which one unit is based on a 99% kill of the standard inoculum.
  • In a typical purification, the CM-cellulose effluent had a relative activity of 37,000 against S. aureus and 24,200 against B. subtilis. Relative activity is the specific activity (beta-lysin units per mg of protein) of the sample divided by that of serum, so these are gains per unit of protein, not in total activity.
  • The authors stated that the staphylocidal relative activity reflects removal of inhibitory substances as well as the degree of purification.
  • In other experiments the highest relative activity against S. aureus ranged from 27,000 to 47,000.
  • Aqueous extracts of platelets did not kill S. aureus.
  • The staphylocidal beta-lysin was inhibited by all the tested phospholipids, anticoagulants (heparin, sodium citrate, sodium oxalate, EDTA) and protein solutions. The beta-lysin against B. subtilis was inhibited only by the phospholipids.
  • In triplicate experiments, the molecular weight of both beta-lysins ranged from 5,500 to 6,000.
  • Purified staphylocidal beta-lysin was completely resistant to heating at 56 C for 30 minutes.
• The authors' conclusion: The authors concluded that the staphylocidal beta-lysin is a distinct bactericidal agent, separate from lysozyme and from the beta-lysin active against B. subtilis.
• Limitations the authors state:
  • The study examined activity only against S. aureus and B. subtilis.
  • The purified preparations contained both types of beta-lysin, which could not be completely separated.

Topics

Medical terms in this study, explained in our Lexicon: Filter pad.

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