Question

Enzyme X is a highly pigmented protein that imparts the characteristic color to certain blue-green algae. It also facilitates a reaction necessary to the survival of this species; we can follow the kinetics of this reaction by measuring the conversion of Substance X to Substance Y at various times during purification.

Given a pure preparation of these algae, and the required supplies and equipment, devise and outline an empirical procedure for purifying Enzyme X.

What is a good indication of purity in your preparation?

The outline of steps to purify Enzyme X should include detail of what the technique is, why that technique is needed, and what the result will be. The reader needs to have enough information so as to be able to do the experiment. Treat the assignment as one that demonstrates your knowledge of biochemical techniques in protein purification.

As for the length of this assignment, you are describing a protocol; therefore, you need to provide the necessary information for the reader to be able to know how, why, and what, as well as the potential outcomes of the steps and the end result.

Answer 1

Cyanobacteria, also known as Blue Green Algae (BGA) are a class of gram negative bacteria which are considered to be the oldest form of life on the earth. They are very unique due to their oxygenic nature of photosynthesis which is very similar to higher plants. Like red algae and cryptomonads, blue green algae also contain Phycobiliproteins which serve as major accessory pigments during photosynthesis.

Phycobiliproteins are large water soluble supramolecular protein aggregates involved in light harvesting in these organisms and may comprise as much as 40%-60% of the total soluble protein in these cells. These brilliantly colored, proteins can be divided broadly into three classes based on their spectral properties: Phycoerythrin (?max ~565nm), Phycocyanin (?max ~620nm), and Allophycocyanin (?max ~650nm). A fourth Phycobiliprotein known as Allophycocyanin B (?max ~670nm) has also been shown to be present in cyanobacteria in low amounts.

The brilliant colors of Phycobiliproteins are mainly due to covalently bound prosthetic groups that are open-chain
tetrapyrrole chromophores bearing A, B, C and D rings named phycobilins. They are either blue colored
phycocyanobilin (PCB), red colored phycoerythrobilin (PEB), yellow colored phycourobilin (PUB), or purple colored phycobiliviolin (PXB), also named cryptoviolin.

These chromophores are generally bound to the polypeptide chain at conserved positions either by one cysteinyl thioester linkage through the vinyl substituent on the pyrrole ring A of the tetrapyrrole or occasionally by two
cysteinyl thioester linkages through the vinyl substituent on both A and D pyrrole rings.

Materials and methods
Cultures were maintained in chemically defined nitrogen free BG-11 media (Stanier et al., 1971)
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