April 27th update

Greetings fellow BioMinds companions!

Sorry for the tardiness in this post, the week has been very demanding and it was troublesome to find time to write this.
Up to the moment the research I have been involved with has changed. I had ordered some sensor sleeves for the Apollo Free Radical Analyzer; but there were logistical errors that resulted in a delay in the delivery and I have been lead to study other effects of the sulfmyoglobin synthesis reaction. Specifically I have been analyzing the stability of the sulfmyoglobin complex. This is an important factor because it is necessary and crucial in order to follow through with crystallization. At the moment there have been delays, including the possible strike on Friday, so I will have to work as speedily as possible in order to have sufficient amounts of data for the conclusion of the semester.

Now, as for the main topic of the post, the BioMinds poster day was a very successful and interesting activity. One thing that I would like to add, before I go in detail, is that I feel that the poster session was TOO short. I’m guessing that the”re were approximately from 90-100 posters on display. One hour for a poster session of that magnitude I feel, and this is in my most humblest opinion, it was very restrictive on this whole experience. I was very interested in the three, preselected posters that I evaluated, but I still wanted to visit dozens of other posters that I found very captivating. Don’t get me wrong, I could’ve done that outside of the allotted time, after 2:00PM, but everybody was picking up and packing up to leave! Perhaps a little more structure was needed so that a higher interaction between the group could’ve been achieved.

As for the three posters:

1) The first poster I visited was by Adriana Dia. This poster was titled Potential Protein-Protein interaction motifs in envelope Glycoprotein of Dengue virus and other Flavivirus. This poster was very well prepared in all the provided requisites, and additionally the student was also very well prepared. One interesting comment that I would like to make is that this whole research experience seems to involve a computer program that simulates Biochemical interactions. The interactions obviously apply to the study of the Dengue virus, but I found it incredibly interesting that they could be simulated at a digital level.

2) The second poster I visited was by Andrea V. Rivera. The title was Understanding the role of phosphorylation in the nonesense-mediated mRNA decay (NMD) pathway. This poster was also very well done and the presenter was very prepared. I can see why she won the ABRCMS best poster award (i think !?). It was interesting to understand a little but of the specialized research that is ocurring in the field of Oncology. I do understand that this project was very dense and complicated, I just wish I had more time in order to capture the best essence of the project.

3) The third and final poster I visited was by Juliany Rivera. The poster’s title was Extremophilic diversity in mangrove’s rhizosphere; a source of putative novel mocroorganisms with biotechnological potential. This poster clearly met with all the criteria that was established in the evaluation sheets. This research seems to have been an attempt to catalog the biodiversity (at a micro level) at the saline areas of sothwestern PR. I found it highly interesting, especially the future applications that could be given to the field of Biotechnology.

The posters displayed at the BioMinds Research Day were very interesting. The population of research students at the UPR proves to be very rich and diverse in knowledge. The experiences did not seem limited to the fields of natural sciences, and it was very rewarding to see all the future applications that could be given to humanity. I would’ve wished to have visited each poster but due to logistical issues I was not able to. Even though this occured, I am sure that the BioMinds community will have a very promising future, in the years that will follow this experience.

February 27th update

Hey everyone

Sorry for this post being a little late, I have the bad habit of saving posts and publishing them on different days! One basically assumes that saving will make it visible but it doesnt!
Up to the moment research in the lab has been slow. I had ordered some new sensor sleeves at the beginning of the semester and they have not yet arrived. So, at the moment I have achieved some progress but not much with regards to this semesters goals. Hopefully by the end of this week or the beginning of the following week, I will have received the materials that I am lacking. Although this has slowed me down, it doesnt mean I havent had my hands full at the moment. With this I mean that, this semester the laboratory has received 11 NEW undergraduate students that will be researching for college credit! For the past month I have been training them in laboratory protocol and I have trained many in the area of sulfmyoglobin. So, all is not lost yet!
Laboratory research has been a very rewarding experience but I am noticing the difficulties and intricacies it implies. From stocking reactives to ensuring the proper functioning of laboratory equipment, a lot of time can be lost in the efforts to make sure that each of these is up to par. I am also preparing to present at PRISM, meaning that my free time is pretty slim at the moment. Since we are supposed to publish an abstract of our research I have decided to publish the abstract that I sent to PRISM. It includes the description of last semester’s research experience, goals and results: It is as goes…

Abstract
Experimentation consisted of a kinetic analysis of the reactivity of hydrogen sulfide with myoglobin. The purpose of such experiment was to determine the formation constant of sulfmyoglobin (k1), a product of the reaction of myoglobin in presence of O2 or H2O2. This was accomplished by studying the kinetics of the reaction at different concentrations of H2S. The oxidation state of the myoglobin was a mixture of metaquo-myoglobin and oxy-myoglobin. The experimental concentrations of H2S were determined with the aid of the Apollo Free Radical Analyzer. The kinetic study consisted of treating equine myoglobin with H2S at the following proportions: 1:1, 1:2, 1:3, 1:4 and 1:5. In each reaction, the concentration of myoglobin remained constant at [15µM] while the [H2S] was altered. The reactions were conducted in presence atmospherical oxygen. The concentrations of two reagents remained constant (Mb and O2). Each reaction was spectrally analyzed for 38min and individual spectra were taken at 2min intervals. Special interest was given to the red region of the spectra (≈616-620nm) since it houses the unique sulfmyoglobin absorption band. Further analysis revealed that the small value of the formation constant (k1) was representative of a slow reaction rate. The value of the formation constant of sulfmyoglobin (k1) was 0.643654004 s-1M-1.

And so it begins…

This semester proves to be an interesting one in the research laboratory. Up to the moment I plan to pick up where I left off last semester with regards to reaction kinetics. Me and my mentor are planning to execute the sulfmyoglobin synthesis reaction under certain parameters, and this will definitely be taking a few saturdays and sundays out of my time. See, the problem is that last semester the kinetic study focused on the sulfmyoglobin synthesis reaction. The oxidation state of myoglobin was a mixture of oxy-mb and met-mb. My first assignment for this semester is to “re-do” the synthesis but I will be adding sodium dithionite to the stock Mb solution. This will help the Mb be in a uniform oxidation state (most importantly being oxy). By doing this I will be able to see if the formation constant is altered significantly.

Once I get that big question out of the way; then I will be studying the kinetics of the sulfmyoglobin synthesis reaction in the presence of hydrogen peroxide. This should yield some interesting results. As for equipment, it looks like I will be wrestling with the Apollo analyzer once more.

I should have more to share by the end of this month.

C-ya soon !

End of Round II ready for Round III

Hello everyone!!

The semester is just about wrapping up, so things are getting a little hectic in the classroom. As for research, I was able to fulfill all my goals that were set for this semester. I successfully calculated the formation constant of sulfmyoglobin in oxygen. According to my mentor, this had not yet been researched by any known colleague of his. I’m a little excited about this, since it means I might be able to publish something next semester (after much honing and polishing of course!). We’ll see, I will certainly let everyone know if it will work out. Next semester I will begin repeating the kinetic experiments, varying the reagents and their respective concentrations.

I would love to explain in full what my experiments consisted of so that there may be a joint understanding of my accomplishments. I was tempted to post my final report to protoporphyrin9 but I don’t think it would be wise to make it open access just yet. But, in a nutshell my experiments consisted on kinetic studies of the synthesis reaction of sulfmyoglobin. A condensed version of this reaction is as follows:

MbFe^{II (a) O2} MbFe^IIO₂ ^{(b) H2S} MbFe^IIO-O^-• + HS• k¹= 0.643654004s^-1M^-1 SMbA + SMbC

Pretty simple eh? This equation displays the reaction with a single initial oxidation state of the iron atom of myoglobin ( +2 ). This oxidation state can vary in accordance to the species that are present in the solution. In my case, I conducted the kinetic study in the presence of two oxidation states of myoglobin ( + 2 and +3 ). Next semester I will want to repeat the experiments with only one oxidation state present. This is much easier to explain with UV/VIS spectra! The reason I am emphasizing this is because there are certain absorption bands that are present at (+2) that are completely different from the absorption bands at (+3).

Now, supposedly there should be little or no difference in my results if I use a single or double oxidation state of myoglobin. Why? Well the formation constant of Mb is actually the slope of a graph of rate constants vs hydrogen sulfide concentration. What does this mean? This means that if my absorptions vary in a uniform way then the slope of the graph would remain generally unaffected. This does not discredit my work this semester because I was able to achieve linear graphs and my formation constant is representative of a slow synthesis reaction (which it is!) . A formation constant less than 0.7M^-1s^-1 ; is considered pretty slow.

Questions and questions that I know can get to be a little confusing, but it does merit the chance to be further analyzed with more time…right? I plan to get right on it in January with no time to waste. I am sufficiently confident to take this experiment and keep running with it, analyzing the variables and different possibilities. But as for the work this semester I would consider it a great contribution to the field of biochemical science. I was able to quantitatively determine the rate and formation constant of a synthesis reaction involving a hemeprotein.

Well that’s all I have to provide for the moment. Next semester proves to be an exciting one.

May everyone have a Happy Thanksgiving, Merry Christmas & Happy New Year.

Catch ya’ll later

Chris

October 25th post

Greetings once again everybody… I once again forgot that saving the post does not necessarily publish it for the public to see it. Oops J!

At the moment research has improved since my last post. I have been working with the Apollo machine and I seem to have worked out its kinks and occasional hiccups. I must admit the whole issue with this piece of equipment has severely delayed the research goals that I had discussed with my mentor at the beginning of the semester. The free radical analyzer is a fragile piece of equipment and it depends on the perfect interconnection of a diverse amount of factors for it to work properly. I wish the Apollo was as manageable as a pH meter or a spectrophotometer. But up to the moment that technology is still in its perfection phase; so I will learn from my mistakes and proceed with my planned goals.

Due to the unforeseen delays in my research, I consider myself to have completed only 50% of my objectives for this semester. At the moment I am beginning my kinetic experiments with the synthesis reaction of sulfmyoglobin. I plan to study the kinetics of five reactions, in these the [myoglobin] will remain unchanged (≈ 15µM). I will conduct the reactions in presence of oxygen and I will not be using hydrogen peroxide as a catalyst. The reactant concentration that I will be altering will be [H₂S]. This way, the proportions of myoglobin to hydrogen sulfide for the five reactions will be as follows: 1:1 1:2 1:3 1:4 1:5

As I previously mentioned, the reaction will be conducted in presence of oxygen and not hydrogen peroxide as I had previously assumed. The concentration of oxygen throughout these reactions will be [oxygen_{sea level}] and will be relatively unchanged. One significant difference when reacting Mb and H₂S in presence of oxygen will be the duration of the reaction. When I did my experiments last semester (in presence of hydrogen peroxide) the reaction lasted a matter of seconds. By using oxygen, the reaction will last 20 – 30 minutes.

The kinetic study I will be conducting will be via UV-Vis spectroscopy. I will be taking a spectrum of the reaction in progress every 3 minutes; for a total of 10 spectra. I will be spending extra hours this November in order to finish my experiments and provide a good amount of data so that I may have contributed effectively this semester to the Biominds program. If I can complete my established goals this semester then I think I would have made a significant contribution to science; since these experiments will aid other scientists when studying the patterns of reactivity of hydrogen sulfide with myoglobin.

September 25th Post :)

Greetings to all my fellow Biominds companions!

Up to the moment research has begun and it has proven to be a little troublesome in some ways. I will get to those issues a little bit later.

I have already begun using the Apollo free radical analyzer and I am currently practicing and familiarizing myself with its usage. This machine has the ability to measure the presence of different free radicals dissolved in solution. The most common ones that are studied are NO^-, O₂, and H₂S.

In my case, I will be using the machine for sole purposes of studying the levels of H₂S. The machine itself functions in a fascinating way. The molecules of H₂S that are dissolved in solution, pass through a membrane that is permeable for H₂S only. Once inside the electrode, the H₂S is oxidized to HS^-; furthermore, for each molecule of H₂S that travels across the membrane, 2 electrons are produced and registered by the electrode. On the computer screen this will register a signal, typically measured in microamperes but that can obviously vary depending on the experiments conditions.

I will photograph the equipment in order to help you visualize what I am describing J    !

Up to the moment I have had 50% (3) of progress in my research. I understand the workings of this complex machine but there are procedures that must be done before starting any experiment. One of these procedures is the preparation of the reactive and buffers that I will be using. Since I am “building off” my research from last semester then all the materials are essentially the same. Some minor variations that must be taken into account are that each reactive must be degasified and purged with nitrogen. This is to ensure that no oxygen is present in the reaction (the sulfhemoglobin reaction in catalyzed by peroxide; this reaction could be catalyzed by oxygen but I will not venture into that just yet!). Usually the buffers are degasified and purged with nitrogen a total of three times.

A problem that I have had is that the permeable membranes that cover the electrodes of the Apollo Free Radical Analyzer have been breaking. Since the membrane is so sensitive; any tear or hole in it will cause the receptor signal to veer off to high levels without stabilizing. This problem has delayed the progress of my experiment; new membrane sleeves have been ordered and should arrive on Monday (hopefully !!!). Once they arrive I will install them on to the H₂S sensor and perhaps get this show on the road J.

Back again!

Vacation-time is over and a new semester is already off to a fast pace!

I regret to say that I did not participate in any research activities during summertime. I spent my vacation studying and preparing for the MCAT (that I passed!). This meant that I had a daily date with various biology/chemistry textbooks.

As for continuing BioMinds research; I had a meeting with my mentor last week. In that meeting we discussed tentative research goals for this semester. Last semester’s research experience concluded with me being able to synthesize the sulfheme protein at various concentrations. The experience, as a whole, provided me with the knowledge and proper “footwork” to be confident and efficient in the laboratory.

This semester I plan to pick up right where I left off in research. I will begin to learn how to measure the levels of the sulfur ion in the sulf-hemeprotein synthesis reaction. Basically I will be repeating the synthesis various times; but I will control and alter the reactant proportions in order to begin studying the kinematics of the reaction at hand. This will allow me to achieve a large amount of quantitative results that will be of great use and insight for future relevant experiments.

As for new equipment, well I will be working mostly with the Apollo Free Radical Analyzer. This machine (that sounds almost mythic) will measure the concentration of the sulfur ion at various intervals of time.

I will provide more information soon! I am a little overworked with juggling between classes and research. I hope to dedicate a lot of time to the lab this week. Hopefully I will be able to provide a more detailed report once things get underway!

April’s Post

Hey again…sorry for the tardiness on this post. I once again forgot that saving the post did not actually publish it to the general public and viewing.

The semester is winding down and much still remains to be done! Despite it having been a fast paced experience, much has been accomplished. I have fully familiarized myself with the synthesis of the sulfmyoglobin complex. This was one of the crucial objectives of my research this semester; since all the future experiments depend on me being able to properly synthesize this hemeprotein derivative, at any concentration whatsoever. I successfully synthesized sulfmyoglobin at various concentrations, from 10μM to 25μM. Although, these concentrations may appear to be miniscule; for purposes of UV-VIS spectroscopy; they are more than enough.

It was very important for me to synthesize sulfmyoglobin depending on the purpose that I was trying to fulfill. In my case UV-VIS spectroscopy required small concentrations; if I used high concentrations, the optical eye of the spectrophotometer would saturate completely, flatlining my spectrum (happened to me more than once!).

Regretfully, I was not able to crystallize sulf-Mb this semester. Why? Well as with every experiment, various pieces of data are needed. In order to crystallize, the constant of molar extinction was needed, including absolute control of the parameters that I had named in my previous posts. So, for the moment crystallization will be held off until the beginning of the autumn 2008 semester.

This experience has been very enriching and academically challenging. One of the hardest things I faced was the issue of time; being able to juggle between classes and research is no easy task. Also, being able to propose the right amount of goals is very difficult. What I mean is that you should take into account how much can be accomplished in the given amount of time. At times one may get anxious and excited about the future goals of their research; careful steps must be taken in order to not jeopardize the safety and proper documentation of all laboratory work. It has been very hard to control my desires to plunge forward into these experiments; although this feeling is good, you need to learn to crawl before learning to crystallize sulfmyoglobin

For that reason, this semester’s research was based solely upon understanding the spectroscopic properties of myoglobin and sulfmyoglobin. I have been able to familiarize myself with the laboratory setting, thus next semester I will be able to begin my first experiments with the sulfmyoglobin hemeprotein.

At the moment I am learning how to measure the levels of the sulfur ion in the sulfMb synthesis reaction. I have not begun experimenting, since I am still learning how to use the Free Radical Analyzer. Hopefully, by the end of this semester I will have begun studying the kinematics of the sulfmyoglobin synthesis reaction; while varying the different levels of hydrogen sulfide added to this. Once I begin concrete experiments; and have concrete data; I will explain this more in detail.

Next semester I hope to begin studying the kinematics of the sulf-Mb synthesis reaction. This will aid me in gaining a quantitative perspective of the sulf Mb reaction. I hope to be able to write more soon!

What’s up?

Up to know, research has been going a bit slow. We have been consulting with some expert crystallographer’s that have been doing this for many years; and apparently the protocol to crystallize any substance is extremely difficult (we already knew that). What I mean is that various parameters need to be taken into account before jumping knee-deep into an experiment of this magnitude. These parameters are:

1. Myoglobin Concentration

2. Myoglobin time of stability (it loves to interact with air !)

3. Ambient temperature

4. pH

5. Isoelectric point (pI)

6. precipitating agent (usually the crystallization is attempted with about fifty different agents)

In order to properly crystallize a substance (protein or any atomic species), it must form a perfect crystal (no in betweens here!). A perfect crystallization will yied a periodical arrangement of amino acids; all coinciding in the same third dimensional plane.

If the experiment is not completed with needed accuracy, an amorphous precipitate is formed. This amorphous precipitate is basically a non periodical arrangement of the amino acids that are being studied. An analogy in order to better understand this can compared to cement construction. An amorphous precipitate would be similar to throwing a bunch of blocks together and using mortar to fill in the cracks. This would definitely produce a structure but it would be highly unstable. A crystal, on the other hand, would be similar to having each cement block placed in perfect order, thus producing a perfect wall.

It would be a terrible shame if the time and money spent into this project was wasted on crystallizing a water atom. Why a waste of time? Well the crystallization experiment usually spans the course of a month. But once we have the formation of crystals, these must be sent off to be studied via x-ray irradiation, and this process can take years!

At the moment I continue to become familiarized with the Bio-inorganic laboratory. I continue to practice the synthesis of sulf-myoglobin (or Hb). In the laboratory ones best friends are the UV-Vis spectrophotometer and the centrifuge. In order to be certain that sulf-myoglobin has been formed; a large band (known as the Soret) appears around 620.0nm. This is one of the principal ways in which we know that the complex has been formed. Another (quick and easy) way is that the solution turns green when we add the H2S. When Hopefully within the next coming weeks we will proceed with the crystallization.

I have been visiting other blogs (all though I haven’t commented on all of them) and I have noticed that the Biominds community is extremely diverse!

For some reason I was only able to comment on one of the assigned blogs because the other two apparently don’t exist? Well when I write the address down, wordpress asks me if I would like to make another blog with such address? Weird eh? Well if anybody wants to try them out… perhaps my computer has already gone crazy or something; here they are:

anyka28.wordpress.com

jtec.wordpress.com

I know free promotion on my blog! No worries guys! I did visit one blog that was one developing improved hip implants. The hips are one of the more critical areas of the body for the elderly; i was very proud to visit somebody who will be undergoing research in this field.

Once my research speeds up I will be posting more soon; including the initial observations of the crystallization experiment.

I wanted to share some sites with you; here is one a BioMinds companion (her name is Pamela Silen-Rivera) shared with me: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-4RC6R7K-3&_user=1671584&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000054082&_version=1&_urlVersion=0&_userid=1671584&md5=b96ee02209ad816fa95d9d5de8f35e2f

Bonne chance tout le monde, je dois aller !

WordPress is a little tricky!

Sorry everybody; I had written the Feb 22nd post but I had forgotten to publish it. I guess I thought that if I simply saved it would appear on the blog. Big oops on my behalf! I guess you can call me the space cadet for now. I really spaced out on that one!